Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthase

ABSTRACT

Glyphosate-tolerant 5-enolpyruvyl-3-phosphosikimate (EPSP) synthases, DNA encoding glyhphosate-tolerant EPSP synthases, plant genes encoding the glyphosate-tolerant enzymes, plant transformation vectors containing the genes, transformed plant cells and differentiated transformed plants containing the plant genes are disclosed. The glyphosate-tolerant EPSP synthases are prepared by substituting an alanine residue for a glycine residue in a conserved sequence found between positions 80 and 120 in the mature wild-type EPSP synthase.

This application is a continuation-in-part of co-pending applicationSer. No. 054,337, filed May 26, 1987, now abandoned.

BACKGROUND OF THE INVENTION

Recent advances in genetic engineering have provided the requisite toolsto transform plants to contain foreign genes. It is now possible toproduce plants which have unique characteristics of agronomicimportance. Certainly, one such advantageous trait is herbicidetolerance. Herbicide-tolerant plants could reduce the need for tillageto control weeds thereby effectively reducing costs to the farmer.

One herbicide which is the subject of much investigation in this regardis N-phosphonomethylglycine. ##STR1## This herbicide is a non-selective,broad spectrum, postemergence herbicide which is registered for use inmore than fifty crops. This molecule is an acid, which dissociates inaqueous solution to form phytotoxic anions. Several anionic forms areknown. As used herein, the name "glyphosate" refers to the acid and itsanions.

Glyphosate inhibits the shikimic acid pathway which provides a precursorfor the synthesis of aromatic amino acids. Specifically, glyphosatecurbs the conversion of phosphoenolpyruvate and 3-phosphoshikimic acidto 5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the enzyme5-enolpyruvyl-3phosphoshikimate synthase.

It has been shown that glyphosate tolerant plants can be produced byinserting into the genome of the plant the capacity to produce a higherlevel of EPSP synthase.

The present invention provides a means of enhancing the effectiveness ofglyphosate-tolerant plants by producing mutant EPSP synthase enzymeswhich exhibit a lower affinity for glyphosate while maintainingcatalytic activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the amino acid sequences for the EPSP synthaseenzymes from E. coli 11303 and E. coli 11303 SM-1.

FIGS. 2A and 2B show the amino acid sequences for EPSP synthase enzymesfrom various plant, bacteria and fungal species.

FIG. 3 shows a plasmid map for co-integrating plant transformationvector pMON316.

FIG. 4 shows the sequence for the CaMV35S promoter, syntheticmulti-linker and NOS3' transcription terminator/polyadenylation signalused in the vectors described herein.

FIG. 5 shows a plasmid map for binary plant transformation vectorpMON505.

FIG. 6 shows a plasmid map for binary plant transformation vectorpMON530.

FIG. 7 shows a diagrammatic representation of the pistil and anther cDNAclones of tomato EPSP synthase.

FIG. 8 shows a diagrammatic comparison of the genomic clones of EPSPsynthase of petunia and arabidopsis.

FIG. 9 shows the steps employed in the preparation of plasmid pMON851.

FIG. 10 shows the steps employed in the preparation of plasmid pMON857.

FIG. 11 shows representative inhibition data for glyphosate-tolerantEPSP synthase versus wild-type EPSP synthases.

FIG. 12 illustrates the glyphosate tolerance of the mutant maize EPSPsynthase of the present invention versus the wild-type maize EPSPsynthase.

STATEMENT OF THE INVENTION

The present invention provides novel EPSP synthase enzymes which exhibitincreased tolerance to glyphosate herbicide. The subject enzymes of thisinvention have an alanine for glycine substitution as describedhereinafter.

The present invention was enabled in part by the discovery of an E. colibacteria carrying an altered EPSP synthase gene. This organism wasobtained in the following manner.

Cells of E. coli ATCC 11303 were transferred to medium A and incubatedat 37° C.

    ______________________________________                                         MEDIUM A                                                                     ______________________________________                                        10X MOPS.sup.1 medium     50     ml                                           50% glucose solution (50 g/100 ml)                                                                      2      ml                                           100 mM aminomethyl phosphonate                                                                          10     ml                                           (sodium salt)                                                                 Thiamine (5 mg/ml) pH 7.4 1      ml                                           100 mM glyphosate (sodium salt)                                                                         10     ml                                           Deionized water to        500    ml                                           10X MOPS medium:                                                              Per 500 ml                                                                    1 M MOPS (209.3 g/l, pH 7.4)                                                                            200    ml                                           1 M Tricine (89.6 g/l, pH 7.4)                                                                          20     ml                                           0.01 M FeSO.sub.4.7H.sub.2 O (278.01 mg/100 ml)                                                         5      ml                                           1.9 M NH.sub.4 Cl (50.18 g/500 ml)                                                                      25     ml                                           0.276 M K.sub.2 SO.sub.4 (4.81 g/100 ml)                                                                5      ml                                           0.5 mM CaCl.sub.2.2H.sub.2 O (7.35 mg/100 ml)                                                           5      ml                                           0.528 M MgCl.sub.2 (10.73 g/100 ml)                                                                     5      ml                                           5 M NaCl (292.2 g/l)      50     ml                                           0.5% L-Methionine (500 mg/100 ml)                                                                       5      ml                                           micronutrients*                                                               *micronutrients in 25 ml H.sub.2 O                                            ZnSO.sub.4 (2.88 mg/ml)   25     μl                                        MnCl.sub.2 (1.58 mg/ml)   250    μl                                        CuSO.sub.4 (1.6 mg/ml)    25     μl                                        CoCl.sub.2 (7.14 mg/ml)   25     μl                                        H.sub.3 BO.sub.3 (2.47 mg/ml)                                                                           250    μl                                        NH.sub.4 Mo.sub. 7 O.sub.24 (3.71 mg/ml)                                                                25     μl                                        ______________________________________                                         .sup.1 MOPS  3N-morpholino-propane-sulfonic acid                         

After a week, a culture was obtained which could grow rapidly in thepresence of high concentrations of glyphosate in the growth medium (10mM or higher). Analysis of the EPSP synthase activity in the extracts ofthis culture and comparison of its glyphosate sensitivity with that ofwild-type E. coli ATCC 11303 revealed that the mutant organism had analtered EPSP synthase. The glyphosate sensitivity of EPSP synthase ofmutant cells was significantly different from that of wild-type cells.This mutant bacterium was designated E. coli 11303 SM-1. The AroA geneencoding EPSP synthase from this mutant bacterium was isolated asfollows.

The DNA from this bacterium was isolated by the method of Marmur (1961).Southern hybridization using E. coli K-12 aroA gene (Rogers et al.,1983) as the probe established that the aroA gene in the mutantbacterium was on a 3.5 Kb BglII-HindIII fragment. This fragment wascloned into the vector pKC7 (Rao, R. N. & Rogers, 1979) and theresulting plasmid was used for transformation of E. coli. Transformedcolonies were screened for their ability to grow in the presence ofglyphosate (Medium A) and were shown to contain the 3.5 Kb BglII-HindIIIinsert by hybridization with the E. coli K-12 aroA gene. This clone wasdesignated pMON9538.

The nucleotide sequence for the mutant E. coli EPSP synthase aroA genewas determined by the method of Sanger (1977) and the correspondingamino acid sequence for the encoded EPSP synthase deduced therefrom.

All peptide structure represented in the present specification andclaims are shown in conventional format wherein the amino group at theN-terminus appears to the left and the carboxyl group at the C-terminusat the right. Likewise, amino acid nomenclature for the naturallyoccurring amino acids found in protein is as follows: alanine (ala;A),asparagine (Asn;N), aspartic acid (Asp;D), arginine (Arg;R), cysteine(Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q), glycine (Gly;G),histidine (His;H), isoleucine (Ile;I), leucine (Leu;L), lysine (lys;K),methionine (Met;M), phenylalanine (Phe;F), proline (Pro;P), serine(Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine (Tyr;Y) andvaline (Val;V).

Amino acid and nucleotide sequences for the above-described mutant andthe wild-type EPSP synthase enzymes of E. coli are shown in FIG. 1. Themutant E. coli EPSP synthase sequence has an alanine for glycinesubstitution at position 96.

FIG. 2 shows the amino acid sequence for EPSP synthase from variousplant, bacteria and fungal species. Inspection of the sequences andalignment to maximize the similarity of sequence reveals a region ofhighly conserved amino acid residues (indicated by the box) in theregion of the E. coli EPSP synthase mutant where the alanine for glycinesubstitution occurred. Indeed, all EPSP synthase enzymes reported in theliterature and in the present specification reveal a glycine at thisposition in this highly conserved region.

Specifically, the glycine residue which is substituted with the alanineresidue in the preparation of the glyphosate-tolerant EPSP synthases ofthe present invention occurs at position 97 in the EPSP synthase ofAspergillus nidulans (Charles et al., 1986); position 101 in the EPSPsynthase of petunia; position 101 in the EPSP synthase of tomato;position 101 in the EPSP synthase of Arabidopsis thaliana; position 104in the EPSP synthase of Glycine max; position 96 in the EPSP synthase ofE. coli K-12 (Duncan et al., 1984) and position 96 in the EPSP synthaseof Salmonella typhimurium (Stalker et al., 1985).

It has been found that the alanine for glycine substitution can beintroduced into this highly conserved region of other wild-type EPSPsynthase enzymes to yield glyphosate-tolerant EPSP synthase enzymes.FIG. 11 shows representative inhibition data for glyphosate-tolerantEPSP synthases of the present invention versus wild-type EPSP synthases.

Hence, in one aspect the present invention provides glyphosate-tolerantEPSP synthase enzymes and a method for producing such enzymes whichcomprises substituting an alanine residue for the second glycine residuein the highly conserved region having the sequence:

    -L-G-N-A-G-T-A-

located between positions 80 and 120 in the mature wild-type EPSPsynthase amino acid sequence. In most cases the above sequence will belocated between positions 90 and 110 in the mature EPSP synthase.

In one embodiment, glyphosate-tolerant EPSP synthase coding sequencesare useful in further enhancing the efficacy of glyphosate-toleranttransgenic plants. Methods for transforming plants to exhibit glyphosatetolerance are disclosed in European Patent Office Publication No.0218571 and commonly assigned U.S. patent application entitled"Glyphosate-Resistant Plants," Ser. No. 879,814 filed July 7, 1986, nowallowed, not as yet patented the disclosures of which are specificallyincorporated herein by reference. The present invention can be utilizedin this fashion by isolating the plant or other EPSP synthase codingsequences and introducing the necessary change in the DNA sequencecoding for EPSP synthase to result in the aforementioned alanine forglycine substitution in the translated EPSP synthase enzyme.

In another aspect, the present invention provides a transformed plantcell and plant regenerated therefrom which contain a plant gene encodinga glyphosate-tolerant EPSP synthase enzyme having the sequence:

    -L-G-N-A-A-T-A-

located between positions 80 and 120 in the mature EPSP synthase aminoacid sequence. In most cases the above sequence will be located betweenpositions 90 and 110 in the mature EPSP synthase. The gene furthercomprises a DNA sequence encoding a chloroplast transit peptide attachedto the N-terminus of the mature EPSP synthase coding sequence, saidtransit peptide adapted to facilitate the import of the EPSP synthaseenzyme into the chloroplast of a plant cell.

Therefore, in yet another aspect the present invention also provides aplant transformation or expression vector comprising a plant gene whichencodes a glyphosate-tolerant EPSP synthase enzyme having the sequence:

    -L-G-N-A-A-T-A-

located between positions 80 and 120 in the mature EPSP synthase aminoacid sequence.

According to still another aspect of the present invention, a process isprovided that entails cultivating such a plant and, in addition,propagating such plant using propagules such as explants, cuttings andseeds or crossing the plant with another to produce progeny that alsodisplay resistance to glyphosate herbicide.

The EPSP synthase sequences shown in FIG. 2 represent a broadevolutionary range of source materials for EPSP synthases. These datademonstrate that EPSP synthase polypeptides from bacterial, fungal andplant material contain the aforementioned conserved region(-L-G-N-A-G-T-A-). However, those skilled in the art will recognize thata particular EPSP synthase may be produced by and isolated from anothersource material which may not have the exact sequence of the conservedregion. Indeed, it has been found that an alanine may be inserted forthe first glycine of the conserved region of petunia EPSP synthase withno attendant changes in glyphosate sensitivity.

Hence, for purposes of the present invention glyphosate-tolerant EPSPsynthase polypeptides produced by substituting an alanine for the secondglycine in a sequence homologous to the sequence (-L-G-N-A-G-T-A-)located between positions 80 and 120 is considered an equivalent of thepresent discovery and therefore within the scope of the invention.

The glyphosate-tolerant EPSP synthase plant gene encodes a polypeptidewhich contains a chloroplast transit peptide (CTP), which enables theEPSP synthase polypeptide (or an active portion thereof) to betransported into a chloroplast inside the plant cell. The EPSP synthasegene is transcribed into mRNA in the nucleus and the mRNA is translatedinto a precursor polypeptide (CTP/mature EPSP synthase) in thecytoplasm. The precursor polypeptide (or a portion thereof) istransported into the chloroplast.

Suitable CTP's for use in the present invention may be obtained fromvarious sources. Most preferably, the CTP is obtained from theendogenous EPSP synthase gene of the subject plant to be transformed.Alternately, one may also use a CTP from an EPSP synthase gene ofanother plant. Although there is little homology between the CTPsequences of the EPSP synthase gene and the ssRUBISCO gene (see e.g.,Broglie, 1983), one may find that non-homologous CTPs may function inparticular embodiments. Suitable CTP sequences for use in the presentinvention can be easily determined by assaying the chloroplast uptake ofan EPSP synthase polypeptide comprising the CTP of interest as describedin Example 18 hereinafter.

Suitable plants for the practice of the present invention include, butare not limited to, soybean, cotton, alfalfa, oil seed rape, flax,tomato, sugar beet, sunflower, potato, tobacco, maize, wheat, rice andlettuce.

Promoters which are known or found to cause transcription of the EPSPsynthase gene in plant cells can be used in the present invention. Suchpromoters may be obtained from plants or viruses and include, but arenot necessarily limited to, the 35S and 19S promoters of cauliflowermosaic virus and promoters isolated from plant genes such as EPSPsynthase, ssRUBISCO genes and promoters obtained from T-DNA genes ofAgrobacterium tumefaciens such as nopaline and mannopine synthases. Theparticular promoter selected should be capable of causing sufficientexpression to result in the production of an effective amount of EPSPsynthase polypeptide to render the plant cells and plants regeneratedtherefrom substantially resistant to glyphosate. Those skilled in theart will recognize that the amount of EPSP synthase polypeptide neededto induce tolerance may vary with the type of plant.

The promoters used in the EPSP synthase gene of this invention may befurther modified if desired to alter their expression characteristics.For example, the CaMV35S promoter may be ligated to the portion of thessRUBISCO gene which represses the expression of ssRUBISCO in theabsence of light, to create a promoter which is active in leaves but notin roots. The resulting chimeric promoter may be used as describedherein. As used herein, the phrase "CaMV35S" promoter includesvariations of CaMV35S promoter, e.g. promoters derived by means ofligation with operator regions, random or controlled mutagenesis,addition or duplication of enhancer sequences, etc.

The mutant EPSP synthase polypeptides of the present invention may beprepared by either polypeptide synthesis or isolation and mutagenesis ofa EPSP synthase gene to produce the above described glyphosate-tolerantmolecule. Since it is foreseen that the greatest utility of the presentinvention is in the preparation of glyphosate-tolerant plants,nucleotide sequences (either cDNA or genomic) encoding theglyphosate-tolerant EPSP synthase can be easily prepared in thefollowing manner.

cDNA Coding Sequences

Total RNA is isolated from the source material which includes, but isnot necessarily limited to, bacteria, fungi and plant tissue. PolyA-mRNAis selected by oligodT cellulose chromatography. A cDNA library is thenprepared using the polyA-mRNA. The cDNA library is then screened using apreviously cloned EPSP synthase sequence or a suitable oligonucleotideprobe. Suitable oligonucleotide probes include probes based on theconserved region having the amino acid sequence (L-G-N-A-G-T-A) orprobes based on the amino acid sequence of other portions of the EPSPsynthase molecule. The cDNA fragments selected by hybridization are thensequenced to confirm that the fragment encodes the EPSP synthase and todetermine the DNA sequence encoding and adjacent to the conserved aminoacid sequence described above.

The EPSP synthase clone is then altered by oligonucleotide mutagenesisto insert the DNA substitution necessary to result in the alanine forglycine substitution in the conserved amino acid sequence(L-G-N-A-G-T-A). The above procedure produces a cDNA sequence whichencodes the glyphosate-tolerant EPSP synthase of the present inventionbased on the wildtype EPSP synthase of the selected source material.This structural coding sequence can be inserted into functional chimericgene constructs and inserted into suitable plant transformation vectorsto be used in preparing transformed plant cells and regenerated plantsusing the methodology described herein.

Genomic EPSP Synthase Clone

Generally it is preferred that the plant tissue from the plant speciesto be transformed also serve as the source material for the DNA codingsequence for the glyphosate-tolerant EPSP synthase of the presentinvention. In this way, one would easily obtain the chloroplast transitpeptide coding sequence from the plant species to be transformed. Insome cases, it may be beneficial to utilize a genomic clone from theplant species which comprises the introns normally found in theendogenous EPSP synthase gene. The general method described above isalso applicable with the exception that the probes are used to screen agenomic DNA library constructed from the selected plant tissue. Detailedexamples better elucidating this preparation of cDNA and genomic DNAglyphosate-tolerant EPSP synthase constructs of the present inventionare provided below.

PREPARATION OF EPSP SYNTHASE PLANT TRANSFORMATION VECTORS I. EPSPSynthase of Petunia

A. Creation of MP4-G Cell Line

The starting cell line, designated as the MP4 line, was derived from aMitchell diploid petunia (see e.g., Ausubel 1980). The MP4 cells weresuspended in Murashige and Skoog (MS) culture media, (GIBCO, GrandIsland, N.Y.) All transfers involved dispensing 10 ml of suspensioncultures into 50 ml of fresh media. Cultivation periods until the nexttransfer ranged from 10 to 14 days, and were based on visual indicationsthat the culture was approaching saturation.

Approximately 10 ml of saturated suspension culture (containing about5×10⁶ cells) were transferred into 50 ml of MS media containing 0.5 mMglyphosate. The sodium salt of glyphosate was used throughout theexperiments described herein. The large majority of cells were unable toreproduce in the presence of the glyphosate. The cells which survived(estimated to be less than 1% of the starting population) were culturedin 0.5 mM glyphosate and transferred to fresh media containingglyphosate every 10 to 14 days.

After two transfers, the surviving cells were transferred into freshmedia containing 1.0 mM glyphosate. After two transfers at 1.0 mM, thesurviving cells were transferred sequentially into 2.5 mM glyphosate,5.0 mM glyphosate, and 10 mM glyphosate.

The MP4-G cells prepared as described-above were substantially shown bya Southern blot assay (Southern, 1975) to have about 15-20 copies of theEPSP synthase gene, due to a genetic process called "gene amplification"(see e.g. Schimke 1982). Although spontaneous mutations might haveoccurred during the replication of any cell, there is no indication thatany mutation or other modification of the EPSP synthase gene occurredduring the gene amplification process. The only known difference betweenthe MP4 and the MP4-G cells is that the MP4-G cells contain multiplecopies of an EPSP synthase gene and possibly other genes located near iton the chromosomes of the cells.

B. Purification and Sequencing of EPSP Synthase Enzymes

Petunia cells from the MP4-G cell line were harvested by vacuumfiltration, frozen under liquid N₂, and ground to a powder in a Waringblender. The powder was suspended in 0.2 M Tris-HCl, pH 7.8, containing1 mM EDTA and 7.5% w/v polyvinyl-polypyrrolidone. The suspension wascentrifuged at about 20,000×gravity for 10 min to remove cell debris.Nucleic acids were precipitated from the supernatant by addition of 0.1volume of 1.4% protamine sulfate and discarded.

The crude protein suspension was purified by five sequential steps (seeMousdale 1984 and Steinrucken 1985) which involved: (1) ammonium sulfateprecipitation; (2) diethylaminoethyl cellulose ion exchangechromatography; (3) hydroxyapatite chromatography; (4) hydrophobicchromatography on a phenylagarose gel; and (5) sizing on a SephacrylS-200 gel.

The purified EPSP synthase polypeptide was degraded into a series ofindividual amino acids by Edman degradation by a Model 470A ProteinSequencer (Applied Biosystems Inc., Foster City, CA), using the methodsdescribed in Hunkapiller 1983a. Each amino acid derivative was analyzedby reverse phase high performance liquid chromatography, as described byHunkapiller 1983b, using a cyanopropyl column with over 22,000theoretical plates (IBM Instruments, Wallingford CT). A partial aminoacid sequence for petunia EPSP synthase is shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        PETUNIA EPSP SYNTHASE SEQUENCES                                               8            9       10      11    12    13                                   ______________________________________                                        Amino Acid:                                                                           Gln      Pro     Ile   Lys   Glu   Ile                                mRNA strand:                                                                          5'-CAP   CCN     AUU   GAP   CAP   AUU                                                         C                 C                                                           A                 A                                  Complementary                                                                         3'-GTQ   GGN     TAA   TTQ   CTQ   TAA                                DNA strand:              G                 G                                                           U                 U                                  Synthetic                                                                     DNA                                                                           Probes:                                                                       EPSP1:  3'-GTQ   GGP     TAP   TTQ   CTQ   TA                                 EPSP2:  3'-GTQ   GGQ     TAP   TTQ   CTQ   TA                                 EPSP3:  3'-GTQ   GGN     TAT   TTQ   CTQ   TA                                 Exact   5'-CAA   CCC     AUU   AAA   GAG   AUU                                mRNA                                                                          Sequence:                                                                     ______________________________________                                    

C. Synthasis of Probes

Using the genetic code, the amino acid sequence indicated in Table 1 wasused to determine the possible DNA codons which are capable of codingfor each indicated amino acid. Using this information, three differentprobe mixtures were created and designated as EPSP-1, EPSP-2, andEPSP-3, as shown in Table 1. In this table, A, T, U, C, and G representthe nucleotide bases: adenine, thymine, uracil, cytosine and guanine.The letters P, Q, and N are variables; N represents any of the bases; Prepresents purines (A or G); Q represents pyrimidines (U, T, or C).

All oligonucleotides were synthesized by the method of Adams 1983.Whenever an indeterminate nucleotide position (P, Q or N) was reached, amixture of appropriate nucleotides was added to the reaction mixture.Probes were labeled 20 pmol at a time shortly before use with 100 μCiγ-[³² P]-ATP (Amersham) and 10 units polynucleotide kinase in 50 mMTris-HCl, pH 7.5; 10 mM MgCl₂, 5 mM DTT, 0.1 mM EDTA, and 0.1 mMspermidine. After incubation for 1 hr. at 37° C., the probes wererepurified on either a 20% acrylamide, 8 M urea gel or by passage over a5 ml column of Sephadex G25 in 0.1 M NaCl, 10 mM Tris-HCl, pH 7.5, 1 mMEDTA.

D. Preparation of mRNA and Preliminary Testing of Probes

(a) Poly-A mRNA

Total RNA was isolated from the MP4 (glyphosate sensitive) and MP4-G(glyphosate resistant) cell lines as described by Goldberg 1981. TotalRNA was further sedimented through a CsCl cushion as described byDepicker 1982. Poly-A mRNA was selected by oligodT cellulosechromatography. The yield of poly-A RNA was 1.1 micrograms (μg) per gramof MP4 cells and 2.5 μg/gm of MP4-G cells.

(b) Gel Processing of RNA

Ten μg of poly-A RNA from the MP4 or MP4-G cell lines were precipitatedwith ethanol and resuspended in 1×MOPS buffer (20 mM MOPS, pH 7.0, 5 mMsodium acetate and 1 mM EDTA, ph 8.0) containing 50% formamide and 2.2 Mformaldehyde. RNA was denatured by heating at 65° C. for 10 min.One-fifth volume of a loading buffer containing 50% glycerol, 1 mM EDTA,0.4% bromophenol blue and 0.4% xylene cyanol was then added. RNA wasfractionated on a 1.3% agarose gel containing 1.1 M formaldehyde untilbromophenol blue was near the bottom. HaeIII-digested φX174 DNA,labelled with ³² P, was run as a size standard. The DNA markersindicated approximate sizes for the RNA bands.

(c) Transfer of RNA to Nitrocellulose

RNA was transferred to nitrocellulose (#BA85, Schleicher & Schuell,Keene, NH) by blotting the gels overnight using 20X SSC (1X SSC is 0.15M NaCl, 0.015 M sodium citrate, pH 7.0) as the transfer buffer. Aftertransfer, filters were air-dried and baked in a vacuum oven for 2-3 hrsat 80° C.

(d) Preliminary Hybridization with Radioactive Probes

Filters were prehybridized in 6×SSC, 10×Denhardt's solution(1×Denhardt's solution is 0.02% ficoll, 0.02% polyvinylpyrrolidone,0.02% bovine serum albumin), 0.5% NP-40, and 200 μg/ml E. coli transferRNA at 50° C. for 4 hrs. Hybridization was carried out in the freshsolution containing 2×10⁶ cpm/ml of either EPSP-1 or EPSP-2 probe for 48hrs at 32° C. The EPSP-3 probe was not tested since it contained a codon(ATA) that is rarely found in the petunia genome. Hybridizationtemperature (32° C.) used in each case was 10° C. below the dissociationtemperature (Td) calculated for the oligonucleotide with the lowest GCcontent in a mixture. The Td of the probe was approximated by theformulate 2° C.×(A+T)+4° C.×(G+C).

(e) Filter Washing

The filters were washed twice for 15-20 min at room temperature in 6×SSCand then for 5 min at 37° C. with gentle shaking. Filters were thenwrapped in plastic film and autoradiographed for 12-14 hrs at -70° C.with two intensifying screens. The filters were then washed again for 5min with gentle shaking at a temperature of 42° C. The filters wereautoradiographed again for 12-14 hrs. The autoradiographs indicated thatthe probe EPSP-1 hybridized to an RNA of approximately 1.9 kb in thelane containing the poly-A RNA from the MP4-G cell line. Nohybridization to this RNA was detected in the lane containing the poly-ARNA from the MP4 cell line. This result was attributed to overproductionof EPSP synthase mRNA by the MP4-G cell line. The probe EPSP-2, whichdiffers from EPSP-1 by a single nucleotide, showed barely detectablehybridization to the 1.9 kb mRNA of the MP4-G cell line but hybridizedstrongly to a 1.0 kb mRNA from both cell lines. However, the 1.0 kb DNAwas not sufficient to encode a polypeptide of 50,000 daltons, and it isbelieved that one of the sequences in the EPSP-2 probe hybridized to anentirely different sequence in the library. These results suggested thatdegenerate probe mixture EPSP-1 contained the correct sequence for EPSPsynthase. This mixture was used in all subsequent degenerate probehybridization experiments.

E. Preparation of λgt 10 cDNA library

(a) Materials Used

AMV reverse transcriptase was purchased from Seikagaku America, Inc.,St. Petersburg, FL; the large fragment of DNA polymerase I (Klenowpolymerase) was from New England Nuclear, Boston, MA; S1 nuclease andtRNA were from Sigma; AcA 34 column bed resin was from LKB,Gaithersburg, MD; EcoRI, EcoRI methylase and EcoRI linkers were from NewEngland Biolabs, Beverly MA; RNAsin (ribonuclease inhibitor) was fromPromega Biotech, Madison, Wisc. and all radioactive compounds were fromAmersham, Arlington, Hts., IL.

The λgt10 vector (ATCC No. 40179) and associated E. coli cell lines weresupplied by Thanh Huynh and Ronald Davis at Stanford University MedicalSchool (see Huynh 1985). This vector has three importantcharacteristics: (1) it has a unique EcoRI insertion site, which avoidsthe need to remove a center portion of DNA from the phage DNA beforeinserting new DNA; (2) DNA ranging in size from zero to about 8,000bases can be cloned using this vector; and, (3) a library can beprocessed using E. coli MA150 cells (ATCC No. 53104) to remove cloneswhich do not have DNA inserts.

(b) cDNA First Strand Synthesis

Poly-A mRNA was prepared as described in section D.(a) above, andresuspended in 50 mM Tris (pH 8.5), 10 mM MgCl₂, 4 mM DTT, 40 mM KC1,500 μM of d(AGCT)TP, 10 μg/ml dT₁₂ -₁₈ primer, and 27.5 units/ml RNAsin.In a 120 μl reaction volume, 70 units reverse transcriptase were addedper 5 μg of poly-A RNA. One reaction tube contained γ-³² P-dCTP (5uCi/120 μl reaction) to allow monitoring of cDNA size and yield and toprovide a first strand label to monitor later reactions. In order todisrupt mRNA secondary structure, mRNA in H₂ O was incubated at 70° C.for 3 min and the tube was chilled on ice. Reverse transcriptase wasadded and the cDNA synthesis was carried out at 42° C. for 60 min. Thereaction was terminated by the addition of EDTA to 50 mM. cDNA yield wasmonitored by TCA precipitations of samples removed at the start of thereaction and after 60 min. Following cDNA synthesis, the cDNA existed asa cDNA-RNA hybrid. The cDNA-RNA hybrid was denatured by heating themixture in a boiling water bath for 1.5 min, and cooled on ice.

(c) Second Strand DNA Synthesis

Single-stranded cDNA was allowed to self-prime for second strandsynthesis. Both Klenow polymerase and reverse transcriptase were used toconvert ss cDNA to ds cDNA. Klenow polymerase is employed first sinceits 3'-5' exonuclease repair function is believed to be able to digestnon-flush DNA ends generated by self-priming and can then extend theseflush ends with its polymerase activity. Reverse transcriptase is usedin addition to Klenow polymerase, because reverse transcriptase isbelieved to be less likely to stop prematurely once it has bound to atemplate strand. The Klenow polymerase reaction was in a final 100 μlvolume excluding enzyme. The reaction mix included 50 mM HEPES, pH 6.9,10 mM MgCl₂, 50 mM KCl, 500 μM of each dNTP and cDNA. To begin thereaction, 20 to 40 units of Klenow polymerase (usually less than 5 μl )were added and the tubes incubated at 15° C. for 5 hrs. The reaction wasterminated by the addition of EDTA to 50 mM. The mix was extracted withphenol and the nucleic acids were precipitated, centrifuged and dried.

The reverse transcriptase reaction to further extend theanti-complementary DNA strand was performed as described for thereaction to originally synthesize cDNA, except dT₁₀ -₁₈ primer andRNAsin were absent, and 32 units of reverse transcriptase were used in a120 μl reaction. The reaction was terminated by the addition of EDTA to50 mM. The mixture was extracted with an equal volume of phenol and thenucleic acid was precipitated, centrifuged and dried.

(d) S1 Nuclease Treatment

200 μl of 2×S1 buffer (1×S1 buffer is 30 mM sodium acetate, pH 4.4, 250mM NaCl, 1 mM ZnCl₂), 175 μl of H₂ O and 525 units of S1 nuclease wereadded to the tubes containing 125 μl of the second strand synthesisreaction product. The tubes were incubated at 37° C. for 30 min and thereaction was terminated by addition of EDTA to 50 mM. The mixture wasextracted with an equal volume of phenol/chloroform (1:1). The aqueousphase was extracted with ethyl ether to remove residual phenol. The DNAwas precipitated with ethanol and air dried.

(e) EcoRI Methylation Reaction

Since the ds cDNAs were copied from a large variety of mRNAs, many ofthe ds cDNAs probably contained internal EcoRI restriction sites. It wasdesired to protect such cleavage sites from EcoRI cleavage, to enablethe use of blunt-ended EcoRI linkers which were subsequently cleavedwith EcoRI to create cohesive overhangs at the termini.

In an effort to prevent the undesired cleavage of internal EcoRI sites,the ds cDNA was methylated using EcoRI methylase. DNA pellets weredissolved in 40 μl of 50 mM Tris pH 7.5, 1 mM EDTA, 5 mM DTT. Four μl of100 uM S-adenosyl-L-methionine and 1 μl (80 units) of EcoRI methylasewere added. Tubes were incubated at 37° C. for 15 min and then at 70° C.for 10 minutes to inactivate the methylase.

It was subsequently discovered that the methylation reaction describedbelow was unsuccessful in preventing EcoRI cleavage at an internal sitewithin the EPSP synthase coding region, apparently because of inactivemethylase reagent. The cleavage of the internal EcoRI site requiredadditional steps to isolate a full-length cDNA, as described below. Toavoid those additional steps, the methylation reagents and reactionconditions should be used simultaneously on the cDNA and on controlfragments of DNA, and protection of the control fragments should beconfirmed by EcoRI digestion before digestion is performed on the cDNA.

(f) DNA Polymerase I Fill-In Reaction

To the tube containing 45 μl of cDNA (prepared as described above) wereadded 5 μl of 0.1 M MgCl₂, 5 μl of 0.2 mM d(ACGT)TP and 10 units of DNApolymerase I. The tube was incubated at room temperature for 10 min. Thereaction was terminated by the addition of EDTA to 25 mM. One microgramof uncut γgt10 DNA was added as a carrier and the mix was extracted withphenol/chloroform (1:1). The nucleic acid in the mix was precipitatedwith ethanol, centrifuged and dried.

(g) Ligation of EcoRI Linkers to Methylated ds cDNA

Approximately 400 pmoles of EcoRI linkers (5'-CGGAATTCCG-3') weredissolved in 9 μl of 20 mM Tris, pH 8.0, 10 mM MgCl₂, 10 mM DTTcontaining 50 μCi of γ-³² P-ATP (5000 Ci/mmole) and 2 units of T4polynucleotide kinase. The oligonucleotides were incubated at 37° C. for30 minutes to allow them to anneal to each other, creatingdouble-stranded, blunt-ended linkers. 2 units of T4 polynucleotidekinase and 1 μl of 10 mM ATP were added and incubated at 37° C. for anadditional 30 min. The linkers were stored at -20° C. The methylated DNApellet was resuspended in tubes containing 400 pmoles of the kinasedlinkers. Ligation of the EcoRI linkers to the methylated DNA was carriedout by adding 1 μl of T4 ligase and incubating the reaction mixture at12-14° C. for 2 days.

(g) Digestion with EcoRI to Create Cohesive Termini

To 11 μl of the reaction production from Section 1.E.(g) above, 10 ml ofa solution containing 50 mM Tris, pH 7.5, 10 mM MgSO₄, 200 mM NaCl wereadded. T4 DNA ligase was heat inactivated by incubation at 70° C. for 10min. Forty units of EcoRI were added and the incubation was carried outat 37° C. for 3 hr. The reaction was terminated by addition of EDTA to50 mM. The sample was clarified by centrifugation and applied to an AcA34 column.

(i) AcA 34 Column Chromatography

Free linkers (those not ligated to ds cDNA) were removed from ds cDNAwith attached linkers, to prevent them from interfering with theinsertion of the desired ds cDNAs into the cloning vectors. AcA 34 resin(a mixture of acrylamide and agarose beads, normally used for sizing)preswollen in 2 mM citrate buffer and 0.04% sodium azide in water, wasadded to the 1 ml mark of a 1 ml plastic syringe plugged with glasswool. The column was equilibrated with 10 mM Tris-HCl pH 7.5, 1 mM EDTA,400 mM NaCl. The ds cDNA mixtures with ligated linkers and free linkers(˜45 μl) was brought to 400 mM NaCl. 1 μl of 0.5% bromophenol blue dye(BPB) was added, and the sample was applied to the column which was runin equilibration buffer at room temperature. Ten 200 μl fractions werecollected. The BPB dye normally eluted from the column in the sixth tubeor later. Tubes 1 and 2 were combined and used as the source of ds cDNAfor cloning.

(j) Assembly of λgt10 clones

The ds cDNA was mixed with 1 μg of EcoRI-cut λgt10 DNA, precipitatedwith ethanol, and centrifuged. After washing the pellet once with 70%ethanol, the DNA pellet was air dried and resuspended in 4.5 μl of 10 mMTris-HCl pH 7.5, 10 mM MgCl₂, 50 mM NaCl. To anneal and ligate the cDNAinserts to the left and right arms of the λgt10 DNA, the mixture washeated at 70° C. for 3 min., then at 50° C. for 15 min. The mixture waschilled on ice and 0.5 μl each of 10 mM ATP, 0.1 M DTT, and sufficientT4 DNA ligase to ensure at least 90% completion were added. The reactionwas incubated at 14° C. overnight, which allowed the insertion of the dscDNA into the EcoRI site of the λgt10 DNA. The resulting DNA waspackaged into phage particles vitro using the method described bySoherer 1981.

(k) Removal of Phages Without Inserts

Insertion of a cDNA into the EcoRI site of λgt10 results in inactivationof the C1 gene. λgt10 phages with inactivated C1 genes (i.e., withinserts) replicate normally in E. coli MA150 cells. By contrast, λgt10phages without inserts are unable to replicate in the MA150 strain of E.coli. This provides a method of removing λgt10 clones which do not haveinserts.

The phages in the library were first replicated in E. coli C600 (M⁺ R⁻)cells which modified the λgt10 DNA to protect it from the E. coli MA150restriction system. A relatively small number of E. coli C600 cells wereinfected and then plated with a 20 fold excess of MA150 (M⁺ R⁺) cells.The primary infection thus occurred in the M⁺ R⁻ cells where all thephages will grow, but successive rounds of replication occurred in theMA150 cells which prevented the replication of phages without inserts.The amplified phage library was collected from the plates, and afterremoval of agar and other contaminants by centrifugation, therecombinant phages were ready to use in screening experiments.

F. Screening of cDNA Library; Selection of pMON9531

Approximately 600 phages (each plate) were spread on 10 cm×10 cm squareplates of solid NZY agar (Maniatis 1982) with 0.7% agarose. Atranslucent lawn of E. coli MA150 cells were growing on the plates.Areas where the phages infected and killed the E. coli cells wereindicated by clear areas called "plaques", which were visible againstthe lawn of bacteria after an overnight incubation of the plates at 37°C. Six plates were prepared in this manner. The plaques were pressedagainst pre-cut nitrocellulose filters for about 30 min. This formed asymmetrical replica of the plaques. To affix the phage DNA, the filterswere treated with 0.5 M NaOH and 2.5 M NaCl for 5 min. The filters werethen treated sequentially with 1.0 M Tris-HCl, pH 7.5 and 0.5 MTris-HCl, pH 7.5 containing 2.5 M NaCl to neutralize the NaOH. They werethen soaked in chloroform to remove bacterial debris. They were thenair-dried and baked under a vacuum at 80° C. for 2 hours, and allowed tocool to room temperature. The filters were then hybridized with ³²P-labelled EPSP-1 probe (2×10⁶ cpm/filter) as described in Section1.D(e) above. After 48 hr of hybridization, the filters were washed in6x SSC at room temperature twice for 20 min and then at 37 ° C. for 5min. These washes removed non-specifically bound probe molecules, whileprobe molecules with the exact corresponding sequence (which was unknownat the time) remained bound to the phage DNA on the filter. The filterswere analyzed by autoradiography after the final wash. After the firstscreening step, seven positively hybridizing signals appeared as blackspots on the autoradiograms. These plaques were removed from the platesand replated on the fresh plates at a density of 100-200 plagues/plate.These plates were screened using the procedure described above. Fourpositively hybridizing phages were selected. DNA was isolated from eachof these four clones and digested with EcoRI to determine the sizes ofthe cDNA inserts. The clone containing the largest cDNA insert,approximately 330 bp, was selected, and designated λE3. The cDNA insertfrom λE3 was inserted into plasmid pUC9 (Vieira 1981), and the resultingplasmid was designated pMON9531.

To provide confirmation that the pMON9531 clone contained the desiredEPSP synthase sequence, the insert was removed from the pMON9531 cloneby digestion with EcoRI. This DNA fragment was then sequenced by thechemical degradation method of Maxam (1977). The amino acid sequencededuced from the nucleotide sequence corresponded to the EPSP synthasepartial amino acid sequence shown in Table 1.

G. Creation of λF7 Genomic DNA Clone

In order to obtain the entire EPSP synthase gene, chromosomal DNA fromthe MP4-G cells line was digested with BamHI and cloned into a phagevector to create a library, which was screened using the partial EPSPsynthase sequence from pMON9531 as a probe.

(a) Preparation of MP4-G Chromosomal DNA Fragments

MP4-G cells were frozen and pulverized in a mortar with crushed glass inthe presence of liquid nitrogen. The powdered cells were mixed with 8ml/g of cold lysis buffer containing 8.0M urea, 0.35M NaCl, 0.05MTris-HCl (pH 7.5), 0.02M EDTA, 2% sarkosyl and 5% phenol. The mixturewas stirred with a glass rod to break up large clumps. An equal volumeof a 3:1 mixture of phenol and chloroform containing 5% isoamyl alcoholwas added. Sodium dodecyl sulfate (SDS) was added to a finalconcentration of 0.5%. The mixture was swirled on a rotating platformfor 10-15 minutes at room temperature. The phases were separated bycentrifugation at 6,000×g for 15 minutes. The phenol/chloroformextraction was repeated. Sodium acetate was added to the aqueous phaseto a final concentration of 0.15 M and the DNA was precipitated withethanol. The DNA was collected by centrifugation, dissolved in 1×TE(10mM Tris-HCl, pH 8.0, 1 mM EDTA) and banded in a CsCl-ethidium bromidegradient. The DNA was collected by puncturing the side of the tube witha 16 gauge needle. The ethidium bromide was extracted withCsCl-saturated isopropanol, and the DNA was dialyzed extensively against1×TE. Approximately 400 μg of DNA was isolated from 12 g of cells.

MP4-G chromosomal DNA (10 μg) was digested to completion with 30 unitsof BamHI in a buffer containing 10 mM Tris, pH 7.8, 1 mM DTT, 10mMMgCl₂, 50 mM NaCl for 2 hours at 37° C. The DNA was extracted withphenol followed by extraction with chloroform and precipitated withethanol. The DNA fragments were suspended in 1×TE at a concentration of0.5 μg/μl.

(b) Cloning of MP4-G Chromosomal DNA Fragments in λMG14

DNA from phage λMG14 (obtained from Dr. Maynard Olson of the WashingtonUniversity School of Medicine, St. Louis, MO) was prepared by the methoddescribed in Maniatis 1982. 150 μg of DNA was digested to completionwith BamHI in a buffer containing 10mM Tris-HCl, pH 7.8, 1 mM DTT, 10 mMMgCl₂, 50 mM NaCl. The completion of the digest was checked byelectrophoresis through 0.5% agarose gel. The phage DNA was thenextracted twice with phenol-chloroform-isoamyl alcohol (25:24:1) andprecipitated with ethanol. The DNA was resuspended in 1×TE at aconcentration of 150 μg/ml. MgClz was added to 10 mM and incubated at42° C. for 1 hr to allow the cohesive ends of λDNA to reanneal.Annealing was checked by agarose gel electrophoresis.

After annealing, DNA was layered over a 38 ml (10-40%, w/v) sucrosegradient in a Beckman SW27 ultracentrifuge tube. The gradient solutionswere prepared in a buffer containing 1 M NaCl, 20 mM Tris-HCl (pH 8.0),5 mM EDTA. 75 μg of DNA was loaded onto each gradient. The samples werecentrifuged at 26,000 rpm for 24 hours at 15° C. in a Beckman SW 27rotor. Fractions (0.5 ml) were collected from the top of the centrifugetube and analyzed for the presence of DNA by gel electrophoresis. Thefractions containing the annealed left and right arms of λDNA werepooled together, dialyzed against TE and ethanolprecipitated. Theprecipitate was washed with 70% ethanol and dried. The DNA was dissolvedin TE at a concentration of 500 μg/ml.

The purified arms of the vector DNA and the BamHI fragments of MP4-G DNAwere mixed at a molar ratio of 4:1 and 2:1 and ligated using T4 DNAligase in a ligase buffer containing 66 mM Tris-HCl, pH 7.5,5 mM MgCl₂,5 mM DTT and 1 mM ATP. Ligation was carried out overnight at 15° C.Ligation was checked by agarose gel eletrophoresis. Ligated phage DNAcarrying inserts of MP4-G DNA were packaged into phage capsids in vitrousing commercially available packaging extracts (Promega Biotech,Madison, WI). The packaged phage were plated in 10 cm×10 cm squareplates of NZY agar in 0.7% agarose at a density of approximately 6000plaques per plate using E. coli C600 cells. After overnight incubationat 37° C., the plaques had formed, and the plates were removed from theincubator and chilled at 4° C. for at least an hour. The agar plateswere pressed against nitrocellulose filters for 30 minutes to transferphages to the filters, and the phage DNA was affixed to the filters asdescribed previously. Each filter was hybridized for 40 hours at 42° C.with approximately 1.0×10⁶ cpm/filter of the 330 bp cDNA insert isolatedfrom the pMON9531 clone, which had been nick-translated, using theprocedure described in Maniatis (1982). The specific activity of theprobe was 2-3×10⁸ cpm/μg of DNA. Hybridization was carried out in asolution containing 50% formamide, 5x SSC, 5x Denhardt's solution, 200μg/ml tRNA and 0.1% SDS. Filters were washed in 1×SSC, 0.2% SDS at 50°C. and autoradiographed. Several positive signals were observed, andmatched with plaques on the corresponding plate. The selected plaqueswere lifts, suspended in SM buffer, and plated with NZY agar. Thereplica plate screening process was repeated at lower densities untilall the plaques on the plates showed positive signals. One isolate wasselected for further analysis and was designated as the λF7 phage clone.

H. Preparation of pMON9543 and pMON9556

The DNA from λF7 was digested (separately) with BamHI, BglII, EcoRI, andHindIII. The DNA was hybridized with a nick-translated EPSP synthasesequence from pMON9531 in a Southern blot procedure. This indicated thatthe complementary sequence from λF7 was on a 4.8 kb BglII fragment. Thisfragment was inserted into plasmid pUC9 (Vieira 1982), replicated,nick-translated, and used to probe the petunia cDNA library, usinghybridization conditions as described in Section 1.(G), using 10⁶ cpmper filter. A cDNA clone with a sequence that bound to the λF7 sequencewas identified, and designated as pMON9543.

DNA sequence analysis (Maxam 1977) indicated that pMON9543 did notcontain the stop codon or the 3' non-translated region of the EPSPsynthase gene. Therefore, the EPSP synthase sequence was removed frompMON9543, nick-translated, and used as a probe to screen the cDNAlibrary again. A clone which hybridized with the EPSP synthase sequencewas identified and designated as pMON9556. DNA sequence analysisindicated that the insert in this clone contained the entire 3' regionof the EPSP synthase gene, including a polyadenylated tail. The 5' EcoRIend of this insert matched the 3' EcoRI end of the EPSP synthase insertin pMON9531. An entire EPSP synthase coding sequence was created byligating the EPSP synthase inserts from pMON9531 and pMON9556.

I. Preparation of pMON546 Vector with CaMV35S/EPSP Synthase Gene

The EPSP synthase insert in pMON9531 was modified by site-directedmutagenesis (Zoller et al, 1983) using an M13 vector (Messing 1981 and1982) to create a BglII site in the 5' non-translated region of the EPSPsynthase gene. The modified EPSP synthase sequence was isolated by EcoRIand BglII digestion, and inserted into vector, pMON530, a binary vectorfor Agrobacterium-based plant transformation to obtain pMON536. The 1.62kb EcoRI-EcoRI fragment from pMON9556 was then inserted into pMON536 toobtain pMON546. Since pMON530 already contained a 35S promoter from acauliflower mosaic virus (CaMV) next to the BglII site, this created achimeric CaMV35S/EPSP synthase gene in pMON546.

pMON530, a derivative of pMON505 carrying the 35S-NOS cassette, wasprepared in the following manner: The CaMV35S promoter was isolated fromthe pOS-1 clone of CM4-184 as an AluI (n 7143)-EcoRI* (n 7517) fragmentwhich was inserted first into pBR322 cleaved with BamHI, treated withKlenow fragment of DNA polymerase I and then cleaved with EcoRI. Thepromoter fragment was then excised from pBR322 with BamHI and EcoRI,treated with Klenow polymerase and inserted into the SmaI site of M13mp8so that the EcoRI site of the mp8 multi-linker was at the 5' end of thepromoter fragment. The nucleotide numbers refer to the sequence ofCM1841 (Gardner et al., 1981). Site directed mutagenesis was then usedto introduce a G at nucleotide 7464 to create a BglII site. The CaMV35Spromoter fragment was then excised from the M13 as a 330 bp EcoRI-BglIIfragment which contains the CaMV35S promoter, transcription initiationsite and 30 nucleotides of the 5' non-translated leader but does notcontain any of the CaMV translational initiators nor the CaMV35Stranscript polyadenylation signal that is located 180 nucleotidesdownstream from the start of transcription (Covey et al., 1981; Guilleyet al., 1982). The CaMV35S promoter fragment was joined to a syntheticmulti-linker and the NOS 3' non-translated region and inserted intopMON200 (Fraley et al., 1985; Rogers et al., 1986) to give pMON316, seeFIG. 3.

Plasmid pMON316 contains unique cleavage sites for BglII, ClaI, KpnI,XhoI and EcoRI located between the 5' leader and the NOS polyadenylationsignals. Plasmid pMON316 retains all of the properties of pMON200. Thecomplete sequence of the CaMV35S promoter, multi-linker and NOS 3'segment is given in FIG. 4. This sequence begins with the XmnI sitecreated by Klenow polymerase treatment to remove the EcoRI site locatedat the 5' end of the CaMV35S promoter segment.

Plasmid pMON530 (see FIG. 6) is a derivative of pMON505 prepared bytransferring the 2.3 kb StuI-HindIII fragment of pMON316 into pMON526.Plasmid pMON526 is a simple derivative of pMON505 in which the SmaI siteis removed by digestion with XmaI, treatment with Klenow polymerase andligation. Plasmid pMON530 retains all the properties of pMON505 and theCaMV35S-NOS expression cassette and now contains a unique cleavage sitefor SmaI between the promoter and polyadenylation signal.

Binary vector pMON505 is a derivative of pMON200 in which the Ti plasmidhomology region, L1H, has been replaced with a 3.8 kb HindIII to Smalsegment of the mini RK2 plasmid, pTJS75 (Schmidhauser & Helinski, 1985).This segment contains the RK2 origin of replication, oriV, and theorigin of transfer, oriT, for conjugation into using the tri-parentalmating procedure (Horsch Klee, 1986).

Referring to FIG. 5, plasmid pMON505 retains all the important featuresof pMON200 including the synthetic multi-linker for insertion of desiredDNA fragments, the chimeric NOS-NPTII'NOS gene for kanamycin resistancedeterminant for selection of E. coli and A. tumefaciens, an intactnopaline synthase gene for facile scoring of transformants andinheritance in progeny and a pBR322 origin of replication for ease inmaking large amounts of the vector in E. coli. Plasmid pMON505 containsa single T-DNA border derived from the right end of the pTiT37nopaline-type T-DNA. Southern analyses have shown that plasmid pMON505and any DNA that it carries are integrated into the plant genome, thatis, the entire plasmid is the T-DNA that is inserted into the plantgenome. One end of the integrated DNA is located between the rightborder sequence and the nopaline synthase gene and the other end isbetween the border sequence and the pBR322 sequences.

Plasmid pMON546 contained (1) the CaMV35S/EPSP synthase gene; (2) aselectable marker gene for kanamycin resistance (Kan^(R)); (3) anopaline synthase (NOS) gene as a scorable marker; and (4) a right T-DNAborder, which effectively caused the entire plasmid to be treated as a"transfer DNA" (T-DNA) region by A. tumefaciens cells.

This plasmid was inserted into A. tumefaciens cells which contained ahelper plasmid, pGV3111-SE. The helper plasmid encodes certain enzymeswhich are necessary to cause DNA from pMON546 to be inserted into plantcell chromosomes. It also contains a kanamycin resistance gene whichfunctions in bacteria.

A culture of A. tumefaciens containing pMON546 and pGV3111-SE wasdeposited with the American Type Culture Collection (ATCC) and wasassigned ATCC accession number 53213. If desired, either one of theseplasmids may be isolated from this culture of cells using standardmethodology. For example, these cells may be cultured with E. coli cellswhich contain a mobilization plasmid, such as pRK2013 (Ditta 1980).Cells which become Spc/Str^(R), Kan^(S) will contain pMON546, whilecells which become Kan^(R), Spc/Str^(S) will contain pGV3111-SE.

GLYPHOSATE-TOLERANT PETUNIA PLANTS

Leaf disks with diameters of 6 mm (1/4 inch) were taken fromsurface-sterilized petunia leaves. They were cultivated on MS104 agarmedium for 2 days to promote partial cell wall formation at the woundsurfaces. They were then submerged in a culture of A. tumefaciens cellscontaining both pMON546 and GV3111-SE which had been grown overnight inLuria broth at 28° C., and shaken gently. The cells were removed fromthe bacterial suspension, blotted dry, and incubated upside down onfilter paper placed over "nurse" cultures of tobacco cells, as describedin Horsch (1980). After 2 or 3 days, the disks were transferred to petridishes containing MS media with 500 μg/ml carbenicillin and 0. 0.1,0.25, or 0.5 mM glyphosate (sodium salt), with no nurse cultures.

Control tissue was created using A. tumefaciens cells containing thehelper plasmid pGV3111-SE and a different plant transformation vector,pMON505, which contained a T-DNA region with a NOS/NPTII/NOS kanamycinresistance gene and a NOS selectable marker gene identical to pMON546,but without the CaMV35S/EPSP synthase gene.

Within 10 days after transfer to the media containing glyphosate,actively growing callus tissue appeared on the periphery of all disks onthe control plate containing no glyphosate. On media containing 0.1 mMglyphosate, there was little detectable difference between the controldisks and the transformed tissue. At 0.25 mM glyphosate, there was verylittle growth of callus from control disks, while substantial growth oftransformed tissue occurred. At 0.5 mM glyphosate, there was no callusgrowth from the control disks, while a significant number of calli grewfrom the transformed disks. This confirms that the CaMV35S/EPSP synthasegene conferred glyphosate resistance upon the transformed cells.

Transformed petunia plants were produced by regeneration from theabove-described transformed leaf disks by the procedure described inHorsch et al (1985). The transformed plants obtained contained thepMON546 vector, described hereinabove, which contains the CaMV 35Spromoter fused to the wild-type- petunia EPSP synthase gene.

Four individual representative transgenic seedlings were selected, grownand tested in the testing procedure described below, along with fourindividual non-transformed (wild-type) petunia seedlings.

The plants were grown in a growth medium in a growth chamber at 26° C.with 12 hours of light per day. The plants were fertilized weekly with asoluble fertilizer and watered as needed. The plants were sprayed at auniform and reproducible delivery rate of herbicide by use of anautomated track sprayer. The glyphosate solution used was measured aspounds of glyphosate acid equivalents per acre, mixed as the glyphosateisopropylamine salt, with an ionic surfactant.

Four individual wild-type (non-transformed) petunia plants were selectedfor use as control plants. Four individual transformed plants containingthe pMON546 vector were selected by kanamycin resistance as described inHorsch et al (1985).

The control plants and the transformed plants were sprayed with theisopropylamine salt of glyphosate at the application level listed inTable 2 below; the experimental results obtained are also summarized inTable 2.

                  TABLE 2                                                         ______________________________________                                        Plant Response to Glyphosate Spraying                                         Plant Type                                                                              Glyphosate Dose*                                                                              Visual Appearance                                   ______________________________________                                        Control.sup.1                                                                           0.8 #/acre      completely dead,                                                              plants showed very                                                            rapid chlorosis and                                                           bleaching, wilted                                                             and died                                            Chimeric EPSP                                                                           0.8 #/acre      growing well,                                       synthase                  slight chlorosis in                                                           new leaves which                                                              are growing with                                                              normal morphology,                                                            plants appear                                                                 healthy and                                                                   started to flower                                   ______________________________________                                         *Acid Equivalent                                                              .sup.1 wildtype plant or transformed with control vector (pMON505)       

As indicated in Table 2, the control plants were killed when sprayedwith 0.8 pounds/acre of glyphosate. In contrast, the petunia plantswhich were transformed were healthy and viable after spraying with 0.8pounds/acre. The transformed plants are more resistant to glyphosateexposure than the non-transformed control plants.

Glyphosate-Tolerant Petunia EPSP Synthase

A plant transformation vector carrying a glyphosate-tolerant petuniaEPSP synthase mutant was prepared in the following manner.

Plasmid pMON342 carries the "mature" wildtype petunia EPSP synthasecoding sequence (without chloroplast transmit peptide) expressed fromthe double phage lambda pL promoter. This plasmid is derived frompMON9544 and pMON9556.

In order to introduce a unique NcoI site and ATG translationalinitiation signal in the wildtype petunia EPSP synthase cDNA justoutside the coding sequence for the mature protein and at the same timeremove the chloroplast transit peptide coding sequence, M8017 (theM13mp9 clone of the 300 bp EcoRI cDNA fragment) was subjected to sitedirected mutagenesis using the procedure of Zoller and Smith (1983) andthe following mutagenesis primer:

    5'-ATCTCAGAAGGCTCCATGGTGCTGTAGCCA-3'

A mutant phage clone was isolated that contained a NcoI site. Thepresence of the above-described mutation was confirmed by sequenceanalysis. This M13mp9 clone was designated M8019.

Plasmid pMON6001 is a derivative of pBR327 (Soberon et al., 1980)carrying the E. coli K12 EPSP synthase coding sequence expressed fromtwo tandem copies of a synthetic phage lambda pL promoter. PlasmidpMON6001 was constructed in the following manner. First, pMON4 (Rogerset al., 1983) was digested with Clal and the 2.5 kb fragment wasinserted into a pBR327 that has also been cleaved with ClaI. Theresulting plasmid, pMON8, contains the EPSP synthase coding sequencereading in the same direction as the beta-lactamase gene of pBR327.

To construct pMON25, a derivative of pMON8 with unique restrictionendonuclease sites located adjacent to the E. coli EPSP synthase codingsequence, the following steps were taken. A deletion derivative of pMON4was made by cleavage with BstEII and religation. The resultant plasmidpMON7 lacks the 2 kb BstEII fragment of pMON4. Next, a 150 bp HinfI toNdeI fragment which encodes the 5' end of the EPSP synthase open readingframe was isolated after digestion of pMON7 with NdeI and HinfI andelectroelution following electrophoretic separation on an acrylamidegel. This piece was added to the purified 4.5 kb BamHI-NdeI fragment ofpMON8 which contains the 3' portion of the EPSP synthase coding sequenceand a synthetic linker with the sequence: ##STR2## The resulting plasmidpMON25 contains the EPSP synthase coding sequence preceded by uniqueBamHI and BglII sites, a synthetic ribosome binding site, and uniqueXbaI and NcoI sites the latter of which contains the ATG translationalinitiator signal of the coding sequence.

To construct pMON6001, pMON25 was digested with BamHI and mixed with asynthetic DNA fragment containing a partial phage lambda pL sequence(Adams and Galluppi, 1986) containing BamHI sticky ends: ##STR3## Theresulting plasmid pMON6001 carries two copies of the synthetic phagelambda pL promoter fragments as direct repeats in the site of pMON25 inthe correct orientation to promote transcription of the EPSP synthasecoding sequence.

Plasmid pMON6001 was cleaved with NcoI and EcoRI and the 3 kb fragmentisolated from an agarose gel. This fragment was mixed with the small 100bp NcoI-EcoRI fragment purified from M8019. Following ligation andtransformation a clone was identified that contained the small 100 bpNcoI-EcoRI fragment corresponding to the 5' end of the "mature" EPSPsynthase of petunia. This construct was designated pMON9544.

Plasmid pMON9544 was digested with EcoRI and treated with alkalinephosphatase. The EcoRI fragment of pMON9544 was mixed with pMON9556 DNAthat had been cleaved with EcoRI to release a 1.4 kb fragment encodingthe 3' portion of the petunia EPSP synthase coding sequence. Followingligation and transformation, a clone was identified that couldcomplement an E. coli aroA mutation and carried the 1.4 kb fragment ofpMON9556. This plasmid was designated pMON342.

The EcoRI site at the 3' end of the EPSP synthase in pMON342 wasreplaced with a ClaI site to facilitate construction. This wasaccomplished by partial digestion with EcoRI followed by digestion withmungbean nuclease to make the ends blunt. ClaI linkers (5'-CATCGATG-3',New England Biolabs) were added to the blunt ends by ligation with T4DNA ligase. The mixture was digested with ClaI to produce sticky ends,and the 5 kb EcoRI partial digest was isolated from an agarose gel andligated with T4 DNA ligase. This plasmid was designated pMON9563.

A 29-nucleotide mutagenic deoxyoligonucleotide having the followingsequence:

    5'-GCCGCATTGCTGTAGCTGCATTTCCAAGG-3'

was synthesized for introducing the alanine for glycine substitution atposition 101 using an automated DNA synthesizer (Applied Biosystems,Inc.). The deoxyoligonucleotide was purified by preparativepolyacrylamide gel electrophoresis.

The 770 bp EcoRI-HindIII fragment of pMON9563 was subcloned into aEcoRI-HindIII digested M13mp10 bacteriophage vector (New EnglandBiolabs). The single-stranded template DNA was prepared from thesubclone as described in the M13 cloning and sequencing handbook byAmersham, Inc. (Arlington Heights, IL.).

Oligonucleotide mutagenesis reactions were performed as described byZoller and Smith (1983). Single-stranded M13mp10 template DNA (0.5picamoles, pmole) containing the 770 bp EcoRI-HindIII fragment of thepMON9563 clone was mixed with 20 pmole of the above-described 29-merdeoxyoligonucleotide and 1 μl of 10×buffer DTT, pH 7.5) in a totalvolume of 10 μl. This mixture was heated at 70° C. for 5 minutes, placedat room temperature (23° C.) for 20 minutes and then placed on ice for20 minutes. During the annealing reaction, the enzyme/nucleotidesolution was prepared by addition of the following components: 1 μl of10×buffer B (0.2M Tris-HCl, 0.1M MgCl₂, 0.1M DTT, pH 7.5), 1 μl each of10 mM dNTPs, 1 μl of 10 mM rATP, 3 units of T4 DNA ligase, 2 units ofthe large fragment of DNA polymerase I and H₂ O to a total volume of 10μl. This solution was kept on ice until used.

After 20 minutes incubation on ice, 10 μl of the enzyme/nucleotidesolution was added to the annealed DNA, mixed and maintained at 15° C.overnight. Three units of T4 DNA ligase were added again to ensurecompletion of the extension reaction to yield closed circular DNAmolecules. This construct was designated M9551. The 770 bp EcoRI-HindIIIfragment of M9551 was inserted into pMON9563 between the EcoRI andHindIII sites, replacing the corresponding wildtype fragment. Thisplasmid was designated pMON9566.

Plasmid pMON530 DNA was digested with BglII and ClaI, to which was addedthe 330 bp BglII-EcoRI EPSP synthase 3' fragment from pMON536 andpurified 1.4 kb EcoRI-ClaI EPSP synthase 5' fragment from pMON9566 andthen treated with T4 DNA ligase. Following transformation a plasmid wasisolated that carried the intact mutant EPSP synthase coding sequence ofpetunia (with the coding sequence for the chloroplast transmit peptide)adjacent to the CaMV35S promoter. This plasmid was designated pMON567.Plasmid pMON567 was inserted into A. tumefaciens cells that containedhelper plasmid pGV3111-SE.

A culture of A. tumefaciens cells containing pMON567/pGV3111-SE wascontacted with leaf disks taken from tobacco plants (Nicotiana tobacamCV H425) as described by Horsch (1985). The Agrobacterium cells insertedthe mutant EPSP synthase gene into the chromosomes of the plant cells.Plant cells resistant to kanamycin were selected and regenerated intodifferentiated plants by the procedure of Horsch (1985).

Progeny of these plants were propagated and grown to a rosette diameterof about 10 cm corresponding to a plant age of about four weeks. Theplants were sprayed with glyphosate at levels corresponding to 2.0 and3.6 pounds acid equiv./acre. The effect of glyphosate on the transformedplants was scored at 7 and 14 days. The effect was translated to anumerical scale of 0-10 in which 0 represents total kill and 10 is thenormal, unsprayed plant. The data below demonstrates that tobacco plantstransformed with the glyphosate-tolerant EPSP synthase gene of petuniaexhibit substantial tolerance even to these high levels of glyphosate.The values represent the best transformant for both wild-type EPSPsynthase and glyphosate-tolerant EPSP synthase genes.

                  TABLE 3                                                         ______________________________________                                        Relative Effect of Glyphosate.sup.1                                           pounds/Acre                                                                   0.4                  2.0           3.6                                        Day     GT.sup.2                                                                             WT.sup.3  GT   WT     GT   WT                                  ______________________________________                                         7      8.0    6.0       8.0  5.0    5.0  5.0                                 14      8.0    7.0       8.3  1.8    7.4  1.7                                 28      9.0    9.0       7.0  0.8    7.0  0.8                                 ______________________________________                                         .sup.1 0 represents total kill and 10 represents no effect.                   .sup.2 Glyphosatetolerant petunia EPSP synthase.                              .sup.3 Wildtype EPSP synthase.                                           

II. EPSP SYNTHASE OF TOMATO

Complementary DNA (cDNA) libraries were prepared from poly-A plus RNAisolated from mature tomato pistils or anthers by a modification of themethods of Huynh et al. (1985) and Gubler et al. (1983) as follows:

First Strand Synthesis

Quantities given below are those used to prepare the mature pistil cDNAlibrary, the anther cDNA library was prepared in a similar manner.

10 μl of 400 μg/ml Actinomycin D (Sigma Chemical) in 50% ethanol wasdried down in each reaction tube in a Savant speed vacuum. The followingreagents were added to this tube (the reagents were added in the ordergiven):

    ______________________________________                                        Vol.    Substance        Final Conc/Amount                                    ______________________________________                                        62 μl                                                                              Autoclaved water to final 100 μl                                   10 μl                                                                              10 X first strand                                                                              see below                                                    buffer                                                                10 μl                                                                              5 mM dNTP        500 μM each                                                                A,C,G,T.sup.1                                        10 μl                                                                              100 μg/ml oligo d(pT)                                                                       1 μg.sup.2                                         2 μl                                                                              RNAsin (30 U/μl)                                                                            60 U.sup.3                                            2 μl                                                                              RNA              ˜1.5 μg                                      3 μl                                                                              Reverse Transcriptase                                                                          40 units.sup.4                                        2 μl                                                                              .sup.32 P-dATP   200 Ci/mMole.sup.5                                   ______________________________________                                         .sup.1 Sigma Chemical, St. Louis, MO.                                         .sup.2 Collaborative Research, Lexington, MA.                                 .sup.3 Promega Biotech, Madison, WI.                                          .sup.4 Life Sciences, St. Petersburg, FL.                                     .sup.5 Amersham, Arlington Heights, IL.                                       The reaction mixture was incubated at 42° C. for 60 min. The           reaction mixture was frozen on dry ice and stored at -20 ° C.     

The quantity of cDNA synthesized was determined to be ˜1.31 μg byprecipitation of a portion of the reaction with trichloroacetic acid andscintillation counting.

Purification of First Strand

Biogel P60 (100-200 mesh, Bio Rad, Richmond, CA), pre-swollen in 10 mMTris-HCl/1 mM EDTA, pH 8.0, (TE) was used to pour a column in asiliconed pasteur pipet plugged with siliconized glass wool (bedvolume=1 ml). The column was washed with several volumes of 1 mM Tris pH7.6/ 0.01 mM EDTA. The column was calibrated by running 90 μl of thissame solution plus 10 μl of column marker buffer (see below) over thecolumn. The void volume was determined by the fraction containing theblue dye. More buffer was added to the column to elute the red dye.

The first strand reaction was extracted twice with an equal volume ofphenol. 0.5 μl 2% bromophenol blue was added to the cDNA and it wasloaded on the column, and the void volume was collected.

Column Marker Buffer:

5% Blue Dextrans (2 M dalton, Sigma)

0.05% Phenol Red (or Bromophenol blue at 0.1%) dissolved in 20 mM TrispH 7-8/ 1 mM EDT

Second Strand Synthesis and Methylation

The first strand was dried to ca. 10 μl in a Savant speed vacuum.

    ______________________________________                                        Vol.     Substance        First Conc./Amount                                  ______________________________________                                        3.8  μl   cDNA             ˜500 ng of first                                                        strand                                          10   μl   10X Sec. Strand Buffer                                                                         1 X                                             0.8  μl   5 mM dNTP        40 μM each                                   81.5 μl   Water            to 100 μl final                                                            volume                                          2    μl   DNA Pol I (NEB)  20 U                                            0.4  μl   E. coli DNA ligase                                                                             2 U                                                          (NEB)                                                            0.5 μl                                                                          RNAase H (BRL)                                                                        1 U                                                              3    μl   32P dCTP         30 uCi                                          1    μl   BSA (1:10 dil of BRL)                                                                          50 μg/ml                                     ______________________________________                                         NEB = New England Biolabs, Beverly, MA                                        BRL = Bethesda Research Labs, Gaithersberg, MD                           

The reaction was incubated at 14° C. for 60 min. then at roomtemperature for 60 min.

The following was added:

0.5 μl 5mM dNTP

1 μl T4 DNA polymerase (NEB)

The reaction was incubated for 30 min. at room temperature.

The following were added:

    ______________________________________                                        1.2 μl  mM S-adenosyl      12 μM                                                   L-methionine (Sigma)                                               1.0 μl  EcoRI Methylase (NEB)                                                                            20 U                                            2.4 μl  0.5 M EDTA         12 mM                                           ______________________________________                                    

5 μl was removed from the reaction and added to 260 ng wild type lambdaDNA (NEB) as control for methylation.

The reactions were incubated at 37° C. for 45 min.

Both the main and test reactions were heated to 68° C. for 10 min. toinactivate enzymes.

Measurements of trichloroacetic acid insoluble counts indicated that˜500 ng of ds cDNA (double stranded cDNA) was produced in the reaction.

    ______________________________________                                        10X Second Strand Buffer:                                                     ______________________________________                                        200    mM       Tris-HCl pH 7.4-7.5                                                                           1 M stock                                     50     mM       MgCl.sub.2      1 M stock                                     1.0    M        KCl             4 M stock                                     100    mM       Ammonium sulfate                                                                              1 M stock                                     1.5    mM       Beta-NAD        150 mM stock                                  ______________________________________                                    

Assay for Completeness of Methylation

The following was added to the heat treated test methylation:

2 μl 100 mM Tris-HCl pH 7.6/100 mM MgCl₂ /1.0 M NaCl

12 μl water

1 μl EcoRI (20 units BRL)

0.5 μl pUC19 (0.5 μg, NEB)

The reaction was incubated for 1 hr. at 37° C.

The products were run on an agarose minigel with undigested pUC19, andlambda digested with EcoRI and HindIII as size markers. The pUC19 in thereaction digested to completion indicating that the EcoRI was workingefficiently, the lambda DNA was completely undigested showing that ithad been protected by the methylation reaction. This shows that themethylase was effective in blocking the EcoRI sites in the cDNA fromdigestion.

ds cDNA Clean Up

The second strand reaction mixture was extracted twice with an equalvolume of phenol, run over a P-60 column as described above and the voidvolume was collected and lyophilized in a Savant speed vacuum. The cDNAwas dissolved in 3 μl 1 mM Tris-HCl pH 7.5/0.01 mM EDTA.

Ligation of Linkers to cDNA

The following was mixed in a microfuge tube:

3 μl ds cDNA (500 ng)

2.5 μl Phosphorylated EcoRI linkers (NEB, 250 ng)

1 μl 10×Ligation buffer

1 μl 10 mM ATP

1.5 μl water (for final vol of 10 μl )

1 μl T4 DNA Ligase (˜400 units NEB)

The reaction was incubated at 14° C. for 12 hr.

10×Ligation Buffer

300 mM Tris-HCl pH 7.6

100 mM MgCl₂

50 mM DTT

Removal of Linkers

The following reagents were added:

2 μl 100 mM Tris-HCl pH 7.6/100 mM MgCl₂ /1.0M NaCl

6 μl water

The reaction was heated to 68° C. for 10 min. to inactivate ligase.

The following reagent was added:

2 μl EcoRI (40 units, NEB)

The reaction wa incubated at 37° C. for 2.5 hr.

The reaction was heated to 68° C. for 10 min. to inactivate EcoRI.

Size Cut cDNA and Separate From Linkers

5 μl of loading buffer was added to the digested cDNA/EcoRI linkerreaction. The sample was electrophoresed on a 0.8% Sea Plaque agarose(FMC Corp., Rockland, MD)/TEA (40 mM Tris-Acetate pH 8.2/1.6 mM EDTA)minigel containing 0.3 μg/ml ethidium bromide. The gel was run at 4 V/cmuntil the bromophenol blue dye had migrated 4 cm. Lambda DNA digestedwith HindIII and EcoRI was used as a size marker. The markers werevisualized by UV fluorescence, and a fragment of gel containing cDNAranging in size from ˜600 bp to greater than 10 kb was removed.

Loading Buffer:

250 mM EDTA pH 7

0.2% Bromophenol blue

50% Glycerol

Purification, Ligation and Packaging

The volume of the gel slice was determined to be ˜500 μl by weighing andassuming a density of 1.0 g/ml. 140 μl of 20 mM Tris-HCl (pH 7.5)/200 mMNaCl/1.0 mM EDTA, and 20 μl of 5 M NaCl were added to the gel fragment.The mixture was heated to 68° C. for 15 min. and extracted twice with500 μl of phenol. The DNA was purified from contaminants bychromatography on an EluTip D column (Schleicher & Schuell, Keen, NH)according to the manufacturers instructions. The final volume was 450μl. The amount of radioactivity in the sample was determined byscintillation counting of an aliquot, and it was determined that 70 ngof cDNA was contained in the eluted volume.

2 μl (2 ug) lambda gt 10 arms (Vector Cloning Systems, San Diego, CA)were added to the cDNA followed by the addition of 2 volumes of coldethanol. The sample was chilled to -80° C. for 15 min. and theprecipitate was pelleted in a microfuge for 15 min. The tube was drainedand rinsed with 200 μl of -20° C. 70% ethanol with caution so as not todisturb the pellet. The pellet was air dried for 30 min.

The following was added:

7.2 μl Water

1 μl 10 X Ligation buffer

1 μl ATP

0.8 μl T4 DNA ligase

The reaction was incubated for 20 hrs at 14° C.

10 x Ligation Buffer

200 mM Tris-HCl pH 7.6

100 mM MgCl₂

50 mM Dithiothreitol (DTT)

One fourth (2.5 μl ) of the ligation reaction was packaged in vitro intophage using Gigapack packaging extracts (Stratagene Cloning Systems, SanDiego, CA) according to the manufacturers instructions. Subsequentplating of the phage showed that this reaction contained 10⁶ recombinantplaque forming units (PFU). Packaging of the entire ligation mix wouldtherefore produce 4×10⁶ PFU. The remainder of the ligation mix wasstored at -20° C. for future use.

Plaque lifts from the two libraries were screened with a ³² P-labeledfragment from pMON6145 containing the complete coding sequence ofpetunia EPSP synthase. pMON6145 is a derivative of plasmid pGEM2(Promega Biotech, Madison, WI) described in the above-referenced andincorporated application S.N. 879,814, which carries a full-length cDNAclone of petunia EPSP synthase. Two hybridizing plaque were isolatedfrom each library. Maps of the inserts of these phages are shown in FIG.7. The large EcoRI fragments of the two pistil clones (P1 and P2) weresubcloned into pUC19 (New England Biolabs), and the small EcoRIfragments were cloned into pUC119 forming plasmids 9591, 9589, 9595 and9596, respectively.

pUC119 is constructed by isolating the 476 bp Hgi AI/Dra fragment ofbacteriophage M13 and making the ends of the fragment blunt with T4 DNApolymerase (New England Biolabs). This fragment is then inserted intopUC19 (Yanisch-Perron et al., 1985) that has been digested with Nde Iand filled in with Klenow DNA polymerase (New England Biolabs). Theresulting plasmid (pUC119) can be used to produce single stranded DNA ifcells harboring the plasmid are infected with a defective phage such asR408 (Stratagene Cloning Systems).

In order to introduce an NcoI site and an ATG translational initiationcodon at the site predicted to be the start of the mature enzyme for invitro expression in E. coli, the 1.6 kb EcoRI/HindIII fragment ofpMON9591 was cloned into EcoRI/HindIII digested M13mp18 (New EnglandBiolabs) producing a phage designated M9568. This clone was mutagenizedwith the oligonucleotide:

    5'-AGCACAATCTCATGGGGTTCCATGGTCTGCAGTAGCC-3'

as previously described. Sequencing confirmed the success of themutagenesis and the resulting phage was designated M9575. The 1.6 kbEcoRI/HindIII fragment of this phage was inserted into EcoRI/HindIIIdigested pMON6140. This plasmid was designated pMON9717. PlasmidpMON6140 is a derivative of pGEMl (Promega Biotech, Madison, WI) whichcarries the same full-length cDNA clone of petunia EPSP synthase asdescribed above for pMON6145.

In vitro transcription and translation of pMON9717 failed to produce anactive enzyme. Subsequent sequencing of the cDNA from which this clonewas prepared (pMON9591) revealed a single nucleotide deletion in thecoding sequence which would result in a frame shift in the codingsequence. The region containing this deletion was replaced by thecorresponding region from pMON9589 by exchanging the 900 bpBamHI/HindIII fragment of pMON9717 with the corresponding fragment ofpMON9589. This plasmid was designated pMON9718. In vitro analysis ofpMON9718 showed it coded for active tomato EPSP synthase.

A vector for high level expression in E. coli was constructed to furthercharacterize the tomato EPSP synthase. The NcoI/HindIII fragment ofpMON9718 containing the coding sequence for tomato EPSP synthase wasinserted into NcoI/HindIII digested pMON5521. This placed the tomatoEPSP synthase coding sequence under the control of the E. coli RecApromoter (Horii et al., 1980; Sancar et al., 1980). This plasmid wasdesignated pMON9719. Plasmid pMON9719 was able to complement the EPSPsynthase deficiency of an E. coli aroA mutant (SR481) demonstrating thesynthesis of active EPSP synthase.

To introduce the alanine for glycine substitution at position 101 of themature tomato EPSP synthase, the wild-type EPSP synthase coding sequencein phage M9568 was mutagenized with the oligonucleotide

    5'-GCCGCATTGCTGTAGCTGCATTTCCAAGG-3'

by the method of Zoller and Smith (1983) as described previously. Thephage was then mutagenized with the oligonucleotide

    5'-AGCACAATCTCATGGGGTTCCATGGTCTGCAGTAGCC-3'

to introduce an NcoI site and translational initiation codon aspreviously described. The resulting construct was designated M9580. TheEcoRI/BamHI fragment of M9580 which contains the region which had beenmutagenized was inserted into pMON9718 which had been digested withEcoRI and BamHI. This plasmid was designated pMON9728. For expression inE. coli the NcoI/HindIII fragment of pMON9728 was inserted intoNcoI/HindIII digested pMON5521 (described above) producing a plasmidwhich was designated pMON9729. Hence, plasmids pMON9719 and pMON9729 aresimilar plasmids in which the wild-type EPSP synthase andglyphosate-tolerant EPSP synthase (gly(101)-ala) of tomato are under thecontrol of the recA promoter of E. coli. E. coli SR481 cells harboringpMON9719 or pMON9729 were grown under conditions which induce theexpression of the RecA promoter. The cells were lysed and the extractswere assayed for EPSP synthase activity.

Specifically, the bacterial cell paste was washed twice with 0.9%saline, suspended in buffer (100 mM Tris-HCl, 5 mM dithiothreitol, 1 mMEDTA, 10% glycerol and 1 mM benzamidine HCl) passed twice through theFrench Pressure Cell at 1000 psi. The cell extract was separated fromthe cells by centrifuging at 15,000×gravity for 10 mins. at 5° C. It wasdesalted using Sephadex G-50 (Pharmacia, Piscataway, New Jersey). Thedesalted extract was assayed for EPSP synthase activity as follows:

To an assay mix (40 μl) containing shikimate-3-phosphate (2 mM),<C-phosphoenolpyruvate (1 mM, 1.2 mCi/mmol), ammonium molybdate (0.1mM), potassium fluoride (5 mM) in 50 mM HEPES-KOH, pH 7, was added 10 μlof the extract and incubated at 25° C. for 2 mins. The reaction wasquenched by addition of 50 μl of 90% ethanol/0.1 M acetic acid, pH 4.5.70 μl of the reaction mixture was loaded on a SynchroPak AX100 HPLCcolumn (0.4×25 cm) and the column eluted with 0.5 M potassium phosphate,pH 5.5 at 1 ml/min. The radioactivity of the eluent was monitored usinga Radiomatic Flo-One Beta Instrument. (Radiomatics, Florida). The EPSPsynthase activity was determined by measurement of the conversion of ¹⁴C-PEP to ¹⁴ C-EPSP synthase, both of which are resolved under the aboveconditions of chromatography. The protein content of the extract wasdetermined by the method of Bradford (Biorad Labs, California). Thespecific activity of the extract is expressed as nanomoles of EPSPsynthase formed/min/mg protein.

Assay results show that cells containing pMON9719 had an EPSP synthaseactivity of about 1600 nanomoles of EPSP synthase formed/minute/mgprotein. However, this enzyme was quite sensitive to glyphosate asindicated by an Iso of 12μM glyphosate. Although cells containingpMON9729 (gly(101)-ala) had a EPSP synthase activity of 390nanomoles/minute/mg protein, this enzyme was highly glyphosate-tolerantas indicated by an I₅₀ value of 32 mM glyphosate.

A plant transformation vector capable of producing the glyphosateresistant form of tomato EPSP synthase in transgenic plants isconstructed as follows:

A BglII site is engineered upstream of the ATG translation initiationcodon of tomato pre-EPSP synthase by performing site directedmutagenesis on pMON9596. The mutagenesis is performed by the method ofKunkel (1985) using the oligodeoxynucleotide:

    5'-GCCATTTCTTGTGAAAAAGATCTTTCAGTTTTTC-3'

The alanine for glycine substitution is engineered into the codingsequence for M9568 by site directed mutagenesis exactly as describedabove. The 700 bp EcoRI/BamHI fragment of the resulting phage is thentransferred into EcoRI/BamHI digested pMON9718 replacing thecorresponding wild-type fragment.

The 70 bp BglII/EcoRI fragment of the altered pMON9596 is then combinedwith the 1.6 kb EcoRI/HindIII fragment of the M9718 derivative intoBglII/HindIII digested pMON550. pMON550 is a derivative of pUC19(Yanisch-Perron et al., 1985) produced by inserting the synthetic DNAfragment: ##STR4## into pUC19 digested with HindIII and Kpn I. Thisreconstitutes a complete tomato EPSP synthase precursor gene whichincludes the alanine for glycine substitution.

For insertion into a plant transformation vector a convenient site isengineered at the 3'-end of the coding sequence by digestion withHindIII, making the ends blunt and inserting a ClaI linker (New EnglandBiolabs). The 1.7 kb BglII/Clal fragment of this plasmid is theninserted into BglII/Cla digested plant transformation vector such aspMON316. The resulting plasmid has the tomato EPSP synthase precursorcoding sequence with the alanine for glycine substitution at position101 of the mature EPSP synthase sequence under control of the CaMV35Spromoter. Transformation of plants, such as tomato, with this vectorleads to the production of a high level of the glyphosate-tolerantenzyme, resulting in glyphosate tolerant plants.

III. EPSP SYNTHASE OF ARABIDOPSIS

An Arabidopsis thaliana genomic bank was prepared by cloning sizefractionated (15-20 kb) MboI partially digested DNA into BamHI and EcoRIdigested lambda EMBL3 (Strategene Cloning Systems, San Diego, CA).Approximately 10,000 plaques of phage from this library were screenedwith ³² P labeled petunia EPSP synthase probe (pMON9566 describedhereinbefore). A strongly hybridizing plaque, designated E1, waspurified. Southern blots of the EPSP synthase probe to the phage DNAidentified two fragments which hybridized very strongly. The firstfragment was a 1.0 kb HindIII fragment and the other was a 700 bp BamHIfragment. These fragments were subcloned into plasmid pUC119 anddesignated pMON574 and pMON578.

The DNA sequences for the two inserts were then determined by the methodof Sanger (1977). The sequence data indicated that the phage did containthe EPSP synthase gene of Arabidopsis by its strong homology to thepetunia EPSP synthase sequence. The 700 bp BamHI fragment was used as ahybridization probe against the phage and Arabidopsis genomic DNA toidentify restriction fragments suitable for the cloning of the entireEPSP synthase gene. Two hybridizing BglII fragments of 6.0 kb and 3.2 kbwere identified in the E1 phage clone. These fragments were separatelysubcloned into pMON550 to provide DNA for further experiments anddesignated pMON582 and pMON583, respectively. Two additional subcloneswere made from clones pMON582 and pMON583. Plasmid pMON584 is the 1.8 kbEcoRI to BamHI fragment containing the 5'-end of the Arabidopsis EPSPsynthase gene in pUC118 which is prepared from pUC18 in a manneranalogous to the preparation of pUC119 from pUC19. Plasmid pMON589 isthe 2.8 kb BamHI to BglII fragment containing the 3'-end of theArabodopsis EPSP synthase gene in pUC119. Sequence determination fromthe BamHI site of pMON584, and from the BamHI site of pMON589 completedthe sequence of the coding regions of the gene, see FIG. 8.

The coding sequence was altered so that the expressed Arabidopsis EPSPsynthase would include the alanine for glycine substitution at position101 of the mature enzyme. Plasmid pMON578 was mutagenized with theoligonucleotide:

    5'-CTTTACCTCGGTAATGCAGCTACAGCAATGCG-3'

by the method of Kunkel (1985). A portion of the resulting plasmid,pMON594, was sequenced to verify the mutation.

A ClaI site is required just upstream of the translational initiationsite for insertion of the Arabidopsis EPSP synthase gene into planttransformation/expression vectors. A 370 bp SnaBI/BamHI fragment ofpMON584 including the translational initiation site and 65 bp of5'-untranslated region was cloned into EcoRV/BamHI digested BluescriptKS (Stratagene Cloning Systems, San Diego, CA) forming pMON9734. Thisfragment can now be removed from pMON9734 with ClaI and BamHI.

The entire Arabidopsis gene was reconstructed for plant transformationexperiments as follows: the 3.0 kb BamHI to BglII fragment containingthe 3' half of the gene was excised from pMON583 and inserted into theunique BamHI site of pMON9734. This plasmid pMON588, has a unique BamHIsite in the middle of the gene. The 800 bp BamHI fragment from pMON594was then inserted into the unique BamHI site of pMON588. This resultingplasmid, pMON598, contains the entire EPSP synthase gene with thealanine for glycine substitution at position 101 of the mature protein.The entire gene was excised from pMON598 as a 3.5 kb ClaI to EcoRIfragment and cloned into ClaI/EcoRI cut pMON857. This new plasmid,pMON600, contains the entire Arabidopsis gene under the transcriptionalcontrol of the CaMV35S promoter and includes the 3' end of the nopalinesynthase gene. The plasmid was then introduced into AgrobacteriumA208ASE containing the disarmed Ti plasmid pTiT37SE.

Plasmid pMON857 was prepared as follows. The DNA coding sequence for atype IV gentamicin-3-Nacetyltransferase (AAC(3)-IV) enzyme was excisedfrom plasmid pLG62 (Gritz and Davies, 1984). The DNA sequence of thisAAC(3)-IV enzyme has been reported in the literature (Brau et al.,1984). Plasmid pMON825 which comprises the DNA containing the openreading frame (ORF) of the AAC(3)-IV enzyme was prepared in thefollowing manner.

A 143 base pair (bp) TaqI fragment spanning the amino terminal portionof the ORF of the AAC(3)-IV gene was excised from pLG62 and cloned intothe AccI site of plasmid pUC8 (Vieira et al., 1982) creating pMON823.Next, a 1316 bp SacI-PstI fragment from pLG62 containing the remainderof the ORF was cloned into pMON823 previously cut with Sacl and PstIendonucleases. This plasmid was designated pMON824. Plasmid pMON824contains the reconstructed coding sequence of the type IVgentamicin-3-N-acetyltransferase with an EcoRI site immediately upstreamof the start of the ORF. A 1300 bp EcoRI fragment containing the entireORF was then excised from pMON824 and cloned into the EcoR site ofpMON530. This plasmid was designated pMON825. Plasmid pMON825 containsthe entire AAC(3)-IV ORF immediately upstream of the NOS 3'transcriptional terminator/polyadenylation signal.

Referring to FIG. 9, plasmid pMON825 was cut with endonucleases SmaI andBglII. The overhangs resulting from the BglII cut were filled bytreatment with Klenow polymerase and the four nucleotide triphosphates.The flush ends were ligated by treatment with DNA ligase and theresulting plasmid designated pMON840. Plasmid pMON840 was cut withendonuclease EcoRI. The overhangs were filled by treatment with Klenowpolymerase and the four nucleotide triphosphates. The flush ends wereligated by treatment with DNA ligase and the resulting plasmiddesignated pMON841. The above procedure removed the BglII, SmaI andEcoRI restriction sites from the chimeric CaMV35S/AAC(3)-IV/NOS gene.

Other restriction sites were removed by site directed mutagenesis in thefollowing manner. The 2170 bp PstI fragment of pMON841 was introducedinto PstI cut pUC119 producing a construct designated pMON843. The EcoRVsite in the CaMV35S promoter sequence was deleted by site directedmutagenesis (Zoller, 1983) using the oligonucleotide:

    5'-TTACGTCAGTGGAAGTATCACATCAATCCA-3'

producing plasmid pMON844. Sequencing confirmed that pMON844 containedthe above-described EcoRV deletion.

The NOS/NPTII/NOS gene in pMON505 was removed by cleavage of pMON505with Stul and HindIII. It was replaced with the 2220 bp EcoRI (Klenowfilled) HindIII fragment from pMON844 containing theCaMV35S/AAC(3)-IV/NOS gene. This plasmid was designated pMON845. It wassubsequently determined by sequence analysis that theCaMV35S/AAC(3)-IV/NOS chimeric gene which is in pMON845 containedextraneous sequence upstream from the start of the CaMV35S promotersequence carried from the NOS promoter of pMON825. The XmaI site at thestart of the CaMV35S promoter of pMON844 was changed to a HindIII bysite directed mutagenesis using the oligonucleotide:

    5'-TGTAGGATCGGGAAGCTTCCCCGGATCATG-3'

producing plasmid pMON849. The EcoRI/HindIII fragment of pMON845(containing the CaMV35S/AAC(3)-IV/NOS gene) was replaced with thesmaller 1900 bp EcoRI/HindIII fragment of pMON849 carrying the samechimeric gene. This plasmid was designated pMON851. Fragments were usedfrom three plasmids to construct a multipurpose cloning vector employingthe CaMV35S/AAC(3)-IV/NOS gene as a selectable marker and containing aCaMV35S/NOS3' cassette. Referring to FIG. 10, the CaMV35S/NOS3' cassettewas prepared from pMON849 and pMON530. Specifically, pMON849 was cutwith BamHI removing the AAC(3)-IV/NOS sequence while leaving the CaMV35Ssequence. The 294 bp BglII/BamHI fragment from pMON530 containing themultilinker and NOS3' was ligated into the BamHI cut pMON849 producingpMON853.

The Tn7 Spc/Str resistance gene was obtained from pMON120. The 1600 bpEcoRI/AraI fragment of pMON120 containing the Spc/Str gene was clonedinto EcoRI and XmaI cut pUC9 producing plasmid pMON854.

Plasmid pMON856 was prepared by ligation of the following threefragments:

Fragment #1: The 7770 bp EcoRI to BclI fragment from pMON851 containingthe CaMV35S/AAC(3)-IV/NOS selectable marker gene, RK2 replicon, pBR322origin of replication, and the right border sequence from plasmid pTiT37of Agrobacterium tumefaciens.

Fragment #2: The 630 bp HindIII to BamHI fragment from pMON853containing the CaMV35S/NOS3' cassette and the multilinker.

Fragment #3: The 1630 bp HindIII to BamHI fragment from pMON854containing the Spc/Str resistance gene.

Extraneous sequence and restriction sites between the selectable markergene and the Spc/Str gene were removed from pMON856 in the followingmanner. Plasmid pMON856 was cut with StuI and XbaI. The XbaI site wasfilled by treatment with Klenow polymerase and the four nucleotidetriphosphates. Subsequent ligation produced plasmid pMON857 which is auseful plant transformation vector containing the CaMV35S/AAC(3)-IV/NOSselectable marker gene.

The Agrobacterium harboring the pMON600 plasmid was used to transformexplants of Brassica napus. Three gentamicin resistant calli wereobtained. All three of these calli were found to be capable of growth on0.5 mM glyphosate, a concentration which kills wild type callus.Proteins were extracted from samples of the calli and were assayed forEPSP synthase activity. All three of the calli contained EPSP synthaseactivity that was resistant to 0.5 mM glyphosate.

These results demonstrate that the calli transformed with pMON600contain a glyphosate resistant form of EPSP synthase. The EPSP synthasein the extracts was further characterized by titrating the resistance toglyphosate. The I₅₀ of the enzyme was found to be 7.5 mM, this isseveral orders of magnitude higher than has been found for any wild typeplant EPSP synthase. Given the differences in the conditions of theassays (plant extracts which still contain the endogenous (wild-type)EPSP synthase versus extracts from overproducing E. coli) this I₅₀ doesnot differ significantly from the values found for the petunia andtomato enzymes carrying the same alanine for glycine substitution (16and 32 mM respectively).

Plants transformed with plasmid pMON600 are produced by transformingstem explants and culturing under selective conditions which favor shootformation over callus formation. The resulting plants will contain theglyphosate-tolerant form of EPSP synthase and will exhibit elevatedtolerance to glyphosate herbicide.

IV. EPSP SYNTHASE OF Glycine max

The cDNA for EPSP synthase of Glycine max was isolated from a libraryconstructed from RNA isolated from Glycine max root tips. The librarywas constructed using the commercially available Amersham cDNA synthesiskit (Amersham Corp., Arlington Hts., IL), and lambda gt10 from VectorCloning Systems (San Diego, CA). The library was screened with an insertfrom pMON578 which contains part of the EPSP synthase gene andhybridizing plaques were isolated and their inserts subcloned intoBluescript plasmids (Vector Cloning Systems, San Diego, CA), and singlestranded phage. The sequence of a portion of one of the cDNA clones(pMON9752, containing a 1600 bp cDNA) was determined. Referring to FIG.2, the protein deduced from the nucleotide sequence has strong homologyto the petunia sequence in the region corresponding to the matureprotein. Notably, amino acids 94-107 of the petunia enzyme are identicalto amino acids 97-110 of the mature Glycine max enzyme (a three aminoacid insertion in the Glycine max relative to the petunia near the aminoterminus is responsible for the difference in numbering, see FIG. 2).

The Glycine max enzyme was altered to change the glycine at position 104(which corresponds to Gly 101 in petunia) to an alanine by site directedmutagenesis using the oligonucleotide:

    5'-AAAGGACGCATTGCACTGGCAGCATTTCCAA-3'

according to the method of Kunkel (1985).

Initial cDNA clones isolated did not contain the complete sequence ofthe amino terminal chloroplast transit peptide of Glycine max EPSPsynthase. The remaining sequence is isolated by screening the cDNAlibrary until a clone containing this region is obtained or by isolatinga clone from a library of Glycine max genomic DNA by hybridization witha Glycine max cDNA clone. For expression in plants a BglII or otherconvenient site is engineered just upstream of the normal translationalinitiation codon as was done in the petunia and tomato sequences. Thisregion is then combined with the cDNA that had been mutagenized tocontain the alanine for glycine substitution at a common HindIII site.The altered gene is then inserted into a plant transformation vectorsuch as pMON530 which places the coding sequence under the control ofthe CaMV35S promoter. The resulting vector is used to produce transgenicplants which exhibit enhanced tolerance to glyphosate herbicide.

Preparation of a Glyphosate Tolerant Maize EPSP Synthase Gene, andGlyphosate Tolerant Maize Cells

Construction of a glyphosate tolerant maize gene

Maize seeds were imbibed for 12 hr in water, the embryos including thescutella were dissected from the seeds and RNA was purified from thismaterial by the method of Rochester et al. (1986). PolyA-mRNA wasisolated from the RNA by chromatography on oligo dT cellulose, and wasused to construct a cDNA library as described previously. The librarywas screened with a ³² -P labelled RNA probe synthesized in vitro frompMON9717 which had been linearized with HindIII. The probe wassynthesized with T7 RNA polymerase (Promega, Madison, WI) according tothe manufacturers instructions. Hybridizing plaques were isolated,replated, and nitrocellulose lifts from the plates were screened withthe same probe. Plaques representing single clones which hybridizedstrongly to the tomato probe were isolated, propagated and used toprepare DNA. Clone lambda-zld was found to contain a 1.8 kb EcoRIinsert. The insert of this phage was subcloned into the EcoRI site ofBluescript KS+(Strategene, San Diego, CA) to form pMON9935. The completesequence of this cDNA clone was determined and is shown in FIG. 2. Tofacilitate future constructions an Xba I site was engineered immediatelyupstream of the first ATG initiation codon of this clone byoligonucleotide mediated mutagenesis by the method of Kunkel using theoligonucleotide:

    5'-TACCAACCATCGGCGTCTAGAGGCAATGGCGGC-3'

producing plasmid pMON9950. pMON9950 was digested with Xba I andreligated to eliminate the 126 bp Xba I fragment at the 5' end of thecDNA forming pMON9951. To produce a coding sequence for a glyphosatetolerant form of maize EPSP synthase pMON9951 was mutated by the methodof Kunkel using the oligonucleotide:

    5'-CTTCTTGGGGAATGCTGCTACTGCAATGCGGC-3'

resulting in pMON9960. This mutagensis will change a GGA codon to a GCTcodon, changing the second glycine residue in the conserved sequence-L-G-N-A-G-T-A- to an alanine in the resulting protein. The glycineresidue is amino acid 163 of the predicted maize preEPSP synthase. Thiswould correspond to amino acid 95-105 of the mature protein depending onthe precise transit peptidase cleavage site which has not beendetermined.

To demonstrate that this alteration produced a glyphosate tolerant formof maize EPSP synthase, protein was produced from pMON9951 and pMON9960by in vitro transcription with T7 RNA polymerase, followed by in vitrotranslation as follows: Plasmid DNA (pMON9951 and pMON9960) containingthe full-length EPSP synthase cDNA was linearized with EcoRI. Thelinearized plasmid DNA was transcribed in vitro with T7 polymeraseessentially as described by Krieg et al., 1984. The standard reactionbuffer contained 40 mM Tris-HCl (pH 7.9), 6 mM MgCl₂, 10 mMdithiothreitol, 2 mM spermidine, 80 U RNAsin ribuonuclease inhibitor,0.5 mM each of ATP, GTP, CTP and UTP, in a final reaction volume of 100μl. The final RNA pellet was resuspended in 20 μl of sterile water andstored at -80° C. A standard translation reaction contained 100 μl ofnuclease-treated rabbit reticulocyte lysate, 5.7 μl of a 19-amino acidmixture (minus methionine at 1 mM each, 5.7 μl of RNA (total RNAtranscripts derived from 0.63 μg of plasmid DNA), 16 μl RNAsin (20U/μl )ribonuclease inhibitor, and 58.3 μl of [³⁵ S] methionine (14-15 mCi/ml).The in vitro translation products were stored frozen at -80° C.

The products of the in vitro translation were then assayed for EPSPsynthase activity as described herein. Referring to FIG. 12, the productof pMON9951 showed easily detectable EPSP synthase activity in theabsence of glyphosate. When the assay was repeated in the presence of1.0 mM glyphosate no activity was detected. In contrast the mutantpre-enzyme product of pMON9960 showed a high level of tolerance toglyphosate, being only slightly inhibited at 1 mM glyphosate, was 25%inhibited at 10 mM glyphosate and still showing detectable activity at100 mM glyphosate.

For expression in maize cells the coding sequence of the glyphosatetolerant mutant form of maize pre-EPSP synthase is excised from pMON9960and inserted between a promoter known to function in maize cells such asthe CaMV35S promoter, and the poly A addition site of the nopalinesynthase gene. In addition an intron such as the first intron of themaize ADH1 gene may be included in the 5'-untranslated region of theexpression unit which may enhance expression of the chimeric gene(Callis, et al. 1987).

This expression unit is then inserted into a vector which includes theneomycin phosphotransferase gene under control of the CaMV35S promoter,or a similar vector with a marker gene that can allow for selection oftransformed maize cells. This vector, or a similar vector using anyother glyphosate resistant coding sequence constructed as described inthe claims and examples of this application is then introduced intomaize cells as described in the following example.

Preparation of Maize Protoplasts

Protoplasts are prepared from a Black Mexican Sweet (BMS) maizesuspension line, BMSI (ATCC 54022) as described by Fromm et al. (1985and 1986). BMSI suspension cells are grown in BMS medium which containsMS salts, 20 g/1 sucrose, 2 mg/1 (2,4-dichlorophenoxy) acetic acid, 200mg/1 inositol, 130 mg/1 asparagine, 1.3 mg/1 niacin, 0.25 mg/1 thiamine,0.25 mg/1 pyridoxine, 0.25 mg/1 calcium pantothenate, pH 5.8. 40 mlcultures in 125 erlenmeyer flasks are shaken at 150 rpm at 26° C. Theculture is diluted with an equal volume of fresh medium every 3 days.Protoplasts are isolated from actively growing cells 1 to 2 days afteradding fresh medium. For protoplast isolation cells are pelleted at200×g in a swinging bucket table top centrifuge. The supernatant issaved as conditioned medium for culturing the protoplasts. Six ml ofpacked cells are resuspended in 40ml of 0.2 M mannitol/50 mM CaC12/10 mMsodium acetate which contains 1% cellulase, 0.5% hemicellulase and 0.02%pectinase. After incubation for 2 hours at 26° C., protoplasts areseparated by filtration through a 60 um nylon mesh screen, centrifugedat 200×g, and washed once in the same solution without enzymes.

Transformation of Maize Protoplasts Using an Electroporation Technique

Protoplasts are prepared for electroporation by washing in a solutioncontaining 2 mM potassium phosphate pH 7.1, 4 mM calcium chloride, 140mM sodium chloride and 0.2 M mannitol. After washing, the protoplastsare resuspended in the same solution at a concentration of 4 X 10E6protoplasts per ml. One-half ml of the protoplast containing solution ismixed with 0.5 ml of the same solution containing 50 micrograms ofsupercoiled plasmid vector DNA and placed in a 1 ml electroporationcuvette. Electroporation is carried out as described by Fromm et al.(1986). As described, an electrical pulse is delivered from a 122 or 245microFarad capacitor charged to 200 V. After 10 minutes at 4° C. and 10min at room temperature protoplasts are diluted with 8 ml of mediumcontaining MS salts 0.3 M mannitol, 2% sucrose, 2 mg/1 2,4-D, 20%conditioned BMS medium (see above) and 0.1% low melting agarose. After 2weeks in the dark at 26° C., medium without mannitol and containingkanamycin is added to give a final concentration of 100 mg/1 kanamycin.After an additional 2 weeks, microcalli are removed from the liquid andplaced on a membrane filter disk above agarose-solidified mediumcontaining 100 mg/1 kanamycin. Kanamycin resistant calli composed oftransformed maize cells appear after 1-2 weeks.

Glyphosate tolerant maize cells

As described by Fromm et al. (1986), transformed maize cells can beselected by growth in kanamycin-containing medium followingelectroporation with DNA vectors containing chimeric kanamycinresistance genes composed of the CaMV35S promoter, the NPTII codingregion and the NOS 3' end. These cells would also be producing theglyphosate tolerant form of EPSP synthase, and would tolerate elevatedlevels of glyphosate.

Alternative methods for the introduction of the plasmids into maize, orother monocot cells would include, but are not limited to, thehigh-velocity microprojectile method of Klein et al. (1987) or theinjection method of de la Pena et al. (1987).

The embodiments described above are provided to better elucidate thepractice of the present invention. It should be understood that theseembodiments are provided for illustrative purposes only, and are notintended to limit the scope of the invention.

REFERENCES

Adams, S.P. and Galluppi, G. R. (1986) Medicinal Research Reviews6:135-170.

Adams, S.P., et al., (1983) J. Amer. Chem. Soc. 05:661.

Ausubel, F., et al., (1980) Plant Mol. Bio. Newsletter 1:26-32.

Brau, et al., Mol. Gen. Genet. 193:179-187 (1984).

Broglie, R. et al., (1983) Bio/Technology 1:55-61.

Callis, J., Fromm, M. and Walbot, V. (1987) Genes and Develop.1:1183-1200.

Charles, G. Keyte, J. W., Brammer, W. J., Smith, M. and Hawkins, A. R.(1986) Nucleic Acids Res. 14:2201-2213.

Covey, S., Lomonosoff, G. and Hill, R., (1981) Nucleic Acids Res.9:6735.

de la Pena, A., Lorz, H. and Schell, J. (1987) Nature 325:274-276.

DePicker A., et al., (1982) J. Mol. Appl. Gen. 1:561.

Ditta, G., et al., (1980) Pro. Natl. Acad. Sci. USA 77:7347.

Duncan, K., Lewendon, A. and Coggins, J. R. (1984) FEBS Lett. 170:59-63.

Fraley, R., Rogers, S., Eichholtz, D., Flick, J., Fink, C., Hoffman, N.and Sanders, P., (1985) Bio/Technology 3:629.

Fromm, M., Taylor, L. P. and Walbot, V. (1985) Proc. Nat Acad. Sci. USA82:5824-5828.

Fromm, M., Taylor, L. P. and Walbot, V. (1986) Nature 319:791-793.

Gardner, R., Howarth, A., Hahn, P., Brown-Luedi, M., Shepherd, R. andMessing, J., (1981) Nucleic Acids Res. 9:2871.

Gritz and Davies, Gene 25:179-188 (1984).

Guilley, H., Dudley, R., Jonard, G., Balax, E. and Richards, K. (1982)Cell 30:763.

Goldberg, R. B., et al., (1981) Devel. Bio. 83:201-217.

Gubler, U., and Hoffman, B. H. (1983) Gene 25:263-269.

Horsch, R. and Klee, H., (1986) Proc. Natl. Acad. Sci. USA vol. 83,4428-4432.

Horsch, R. B., and Jones, G. E. (1980) In Vitro 16:103-108.

Horii, T., et al. (1980) P.N.A.S. USA 77:313.

Hunkapiller, M. W., et al. (1983b) Methods Enzymol. 91:486-493.

Huynh, T.V. Young, R.A. and Davis, R.W. (1985) DNA Cloning Techniques: APractical Approach, D. Glover ed., IRL Press, Oxford.

Klein, T.M., Wolf, E.D., Wu, R. and Sanford, J.C. (1987) Nature327:70-73.

Krieg, P.A. and Melton, D.A. (1984) Nucleic Acids Res. 12:7057-7070.

Kunkel (1985) P.N.A.S. USA 82:488-492.

Lemke, G. and Axel, R. (1985) Cell 40:501-508.

Maniatis, T., et al. (1982) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Labs., NY.

Marmur, J. (1961) J. Mol. Biol. 3:208-210.

Maxam, A.M. and Gilbert, W., (1977) P.N.A.S. USA 74:560-564.

Messing, J. et al. (1981) Nucleic Res. 9:309-321.

Mousdale, D.M. and Doggins, J.R. (1984) Planta 160:78-83.

Okayama, H. and Berg, P. (1982) Mol. Cell. Biol. 2:161.

Rao, R.N. and Rogers, S.G. (1979) Gene pp. 7:79-82.

Rochester, D.E., Winter, J.A. and Shah, D.M. (1986) EMBO J. 5:452-458.

Rogers, S., Horsch, R. and Fraley, R., (1986) Methods in Enzymology Vol.118 (H. Weissbach and A. Weissbach, eds.) p.627, Academic Press, NewYork.

Rogers, S.G., et al. (1983) Appl. Envir. Microbio. 46:37-43.

Sancar, A., et al. (1980) P.N.A.S. USA 77:2611.

Sanger, et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463.

Scherer, et al. (1981) Developmental Bio. 86:438-447.

Schimke, R.T. ed. (1982) Gene Amplification, Cold Spring Harbor Labs.

Schmidhauser, T. and Helinski, D. (1985) J. Bacteriology 164:446.

Soberson, et al. (1980), Gene 9:287-305.

Southern, E.M. (1975) J. Mol. Biol. 98:503-517.

Stalker, D. M., Hiatt, W. R. and Comai, L. (1985) J. Biol Chem.260:4724-4728.

Steinrucken, H. and Amrhein, N. (1980) Biochem. & Biophys. Res. Comm.94:1207-1212.

Vieira, J. et al., (1982) Gene 19:259-268.

Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene 33:103-119.

Zoller, M.M. et al. (1983) Methods Enzymol. 100:468.

We claim:
 1. A method for producing a gene encoding aglyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate (EPSP) synthaseenzyme which comprises altering a gene encoding EPSP synthase to causethe substitution of an alanine residue for the second glycine residue inthe amino acid sequence:

    -L-G-N-A-G-T-A-

located between positions 80 and 120 in a mature EPSP synthase sequence.2. A method of claim 1 in which the glyphosate-tolerant EPSP synthasegene is produced from a plant EPSP synthase gene.
 3. A method of claim 1in which the glyphosate-tolerant EPSP synthase gene is produced from abacterial EPSP synthase gene.
 4. A method of claim 1 in which theglyphosate-tolerant EPSP synthase gene is produced from a fungal geneencoding EPSP synthase.
 5. A glyphosate-tolerant5-enolpyruvyl-3-phosphoshikimate (EPSP) synthase enzyme which containsthe amino acid sequence:

    -L-G-N-A-A-T-A-

between positions 80 and 120 in the mature EPSP synthase sequence. 6.The glyphosate-tolerant EPSP synthase enzyme of claim 5 as shown inFIGS. 1(a) and 1(b).
 7. A glyphosate-tolerant EPSP synthase geneproduced by the method of claim 1 wherein the wild-type EPSP synthasecoding sequence is selected from the group of EPSP synthases consistingof Aspergillus nidulans, petunia, tomato, maize, Arabidopsis thaliana,Glycine max, E. coli K-12 and Salmonella typhimurium as shown in FIG. 2.8. A glyphosate-tolerant EPSP synthase gene of claim 7 wherein thewild-type EPSP synthase coding sequence is selected from Aspergillusnidulans.
 9. A glyphosate-tolerant EPSP synthase gene of claim 7 whereinthe wild-type EPSP synthase coding sequence is selected from petunia.10. A glyphosate-tolerant EPSP synthase gene of claim 7 wherein thewild-type EPSP synthase coding sequence is selected from tomato.
 11. Aglyphosate-tolerant EPSP synthase gene of claim 7 wherein the wild-typeEPSP synthase coding sequence is selected from maize.
 12. Aglyphosate-tolerant EPSP synthase gene of claim 7 wherein the wild-typeEPSP synthase coding sequence is selected from Arabidopsis thaliana. 13.A glyphosate-tolerant EPSP synthase gene of claim 7 wherein thewild-type EPSP synthase coding sequence is selected from Glycine max.14. A glyphosate-tolerant EPSP synthase gene of claim 7 wherein thewild-type EPSP synthase coding sequence is selected from E. coli K-12.15. A glyphosate-tolerant EPSP synthase gene of claim 7 wherein thewild-type EPSP synthase coding sequence is selected from Salmonellatyphimurium.